Dual resonance energy transfer nucleic acid probes

ABSTRACT

Dual nucleic acid probes with resonance energy transfer moieties are provided. In particular, fluorescent or luminescent resonance energy transfer moieties are provided on hairpin stem-loop molecular beacon probes that hybridize sufficiently near each other on a subject nucleic acid, e.g. mRNA, to generate an observable interaction. The invention also provides lanthanide chelate luminescent resonance energy transfer moieties on linear and stem-loop probes that hybridize sufficiently near each other on a subject nucleic acid to generate an observable interaction. The invention thereby provides detectable signals for rapid, specific and sensitive hybridization determination in vivo. The probes are used in methods of detection of nucleic acid target hybridization for the identification and quantification of tissue and cell-specific gene expression levels, including response to external stimuli, such as drug candidates, and genetic variations associated with disease, such as cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 10/179,730 filed on Jun. 25, 2002, now U.S. Pat.No. 7,081,336, which claims the priority benefit of U.S. ProvisionalPatent Application Ser. No. 60/300,672 filed Jun. 25, 2001 and U.S.Provisional Patent Application Ser. No. 60/303,258 file by Jul. 3, 2001,the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to the detection of target nucleicacids, such as mRNA. More specifically, the present invention relates toa dual molecular beacons approach that uses resonance energy transfer tosignificantly reduce false-positive signal in detection of targetnucleic acids associated with disease.

BACKGROUND OF THE INVENTION

The ability to monitor and quantify the level of gene expression inliving cells in real time can provide important information concerningthe production, temporal and spatial processing, localization, andtransport of specific mRNA in different conditions. This new type ofinformation could potentially revolutionize biological studies and mayalso have applications in medical diagnostics and therapeutics.Technologies currently available for analysis and quantification of geneexpression such as real-time RT-PCR, Northern blotting, expressedsequence tag (EST), serial analysis of gene expression (SAGE) and DNAmicroarrays are powerful tools for in vitro studies; however, they arenot capable of quantifying gene expression in living cells. There is aclear need to develop molecular probes that can recognize target mRNA inliving cells with high specificity and instantaneously convert suchrecognition into a measurable signal with a high signal-to-backgroundratio.

Molecular beacons are a class of fluorescence-quenched nucleic acidprobes that can be used in a quantitative fashion; these probesfluoresce upon target recognition (i.e., hybridization) with potentialsignal enhancement of >200 under ideal conditions. Structurally, theyare dual-labeled oligonucleotides with a reporter fluorophore at one endand a dark quencher at the opposite end (Tyagi and Kramer; 1996). Theyare designed to have a target-specific probe sequence positionedcentrally between two short self-complementary segments which, in theabsence of target, anneal to form a stem-loop hairpin structure thatbrings the fluorophore in close proximity with the quencher. In thisconfiguration the molecular beacon is in the “dark” state (Bernacchi andMely, 2001). The hairpin opens upon hybridization with a complementarytarget, physically separating the fluorophore and quencher. In thisconfiguration the molecular beacon is in the “bright” state. Transitionbetween dark and bright states allows for differentiation between boundand unbound probes and transduces target recognition into a fluorescencesignal (Matsuo, 1998; Liu et al., 2002).

Linear fluorescent probes, as are used in fluorescence in-situhybridization (FISH) (Femino et al., 1998), are “bright” in both thebound and unbound state. To detect positive signal after hybridization,unbound probe must be removed by washing, which prevents the applicationof this method to gene detection in living cells. In theory, molecularbeacons do not require a washing step and so should be directly usablein living cells (Matsuo, 1998; Sokol et al., 1998). However, interactionbetween molecular beacons and certain intracellular factors can causefluorescence in the absence of target hybridization and lead tofalse-positive signals (Mitchell, 2001). Using conventionalmolecular-beacon-based methods, the fluorescent signal that results fromtarget hybridization cannot be distinguished from any other event thatspatially separates reporter from quencher, such as probe degradation byintracellular nucleases or interaction with DNA binding proteins thatunwind the hairpin stem structure (Li et al., 2000; Dirks et al., 2001;Molenaar, et al., 2001; Fang et al., 2000).

Two linear oligonucleotide probes labeled respectively with donor andacceptor fluorophores have been used in FRET-based studies of DNAhybridization, DNA secondary structure and RNA synthesis (Cardullo etal., 1988; Morrison and Stols, 1993; Sixou et al., 1994; Sei-Iida etal., 2000; Tsuji et al. 2000; Tsuji et al. 2001), however, thesensitivity of intracellular gene detection using such probes suffersfrom strong background signal due to unbound probes and cellautofluorescence.

The unique target recognition and signal transduction capabilities ofmolecular beacons have led to their application in many biochemical andbiological assays including quantitative PCR (Vogelstein and Kinzler,1999; Chen and Mulchandani, 2000), protein-DNA interactions (Fang etal., 2000; Li et al., 2000), multiplex genetic analysis (Marras et al.,1999; de Baar et al., 2001), and the detection of mRNA in living cells(Matsuo 1988; Sokol et al., 1998; Molenaar 2001). However,false-positive signals due to protein-beacon interaction andnuclease-induced beacon degradation significantly limit the sensitivityof the in vivo applications (Mitchell, 2001). The thermodynamic andkinetic properties of molecular beacons are dependent on its structureand sequence in complex ways (Bonnet et al. 1999; Kuhn et al., 2002).Moreover, the signal-to-background ratio in target detection isdependent not only on design (length and sequence of the stem and probe)but also on the quality of oligonucleotide synthesis and purification(Goddard et al., 2000; Bonnet et al., 1998) and the assay conditionsemployed.

Therefore, there is a strong need in the art to provide improvedcompositions and methods for improved detection of nucleic acids thatexhibit high specificity and sensitivity. Furthermore, there is a needfor such compositions and methods that can be used for detection ofgenetic transcription in vivo. There is a need for such improvedcompositions and methods for observing changes in genetic expressionlevels in response to external stimuli, or for the detection of geneticabnormalities indicating a potential or actual disease state.

SUMMARY OF THE INVENTION

The present invention provides compositions for the detection of asubject nucleic acid comprising a first nucleic acid probe thathybridizes to a first nucleic acid target sequence on the subjectnucleic acid, forms a stem-loop structure when not bound to the firstnucleic acid target sequence, and incorporates a resonance energytransfer donor moiety. This embodiment of the invention further providesa second nucleic acid probe that hybridizes to a second nucleic acidtarget sequence on the subject nucleic acid, forms a stem-loop structurewhen not bound to the second nucleic acid target sequence, andincorporates a resonance energy transfer acceptor moiety. The inventionprovides that the first nucleic acid target sequence and the secondnucleic acid target sequence are separated by a number of nucleotides onthe subject nucleic acid such that a resonance energy transfer signalfrom interaction between the donor moiety of the first nucleic acidprobe and the acceptor moiety of the second nucleic acid probe can bedetected to determine hybridization of both the first nucleic acid probeand the second nucleic acid probe to the subject nucleic acid.

Therefore, according to the present invention, alternative compositionsand methods are provided for the detection of subject nucleic acidsequences of interest in a sample. In particularly preferredembodiments, fluorescent or luminescent resonance energy transfermoieties are provided on hairpin stem-loop molecular beacon probes thathybridize sufficiently near each other on a subject nucleic acid, e.g.mRNA, to generate an observable interaction. The invention therebyprovides signal of energy transfer for rapid, specific and sensitivehybridization detection that can be advantageously used in vivo. Theprobes are useful in methods of detection of target nucleic acidhybridization and the identification of genetic expression and thepresence of genetic variations associated with disease, such as cancer.

Accordingly, it is an object of the present invention to provideimproved compositions and methods of use for more sensitive, specific orrapid nucleic acid detection.

It is a further object of the present invention to provide improvedmethods for the detection of gene expression associated with a responseto external stimuli, e.g. a therapeutic drug candidate.

It is a further object of the present invention to provide improvedmethods for the detection of gene expression, including geneticexpression associated with a disease state.

It is another object of the present invention to provide for the use ofsuch compositions and method in vivo.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows hybridization of the donor and acceptor molecular beaconsto adjacent regions on the same mRNA target results in FRET. Bydetecting FRET, fluorescence signals due to probe/target binding can bedistinguished from that due to beacon degradation and non-specificinteractions. In the figure, letters Q, D and A represent respectivelyquencher, donor dye and acceptor dye molecules.

FIG. 2 shows a schematic of the assay system with 4-base spacing betweendonor and acceptor molecular beacons when hybridized to the synthetictarget. In this example both beacons have a probe length of 19 bases anda stem length of 5 bases. The underscores indicate the 38-base sequenceof the target complementary to the beacons. Note that for each beaconone arm of the stem is part of the probe sequence so that the movementof the dye molecules is restricted after hybridization.

FIG. 3 shows typical emission spectra of dual FRET molecular beacons.Three signal-to-background ratios are defined: S:B_(dMB) represents theenhancement in fluorescence of a conventional molecular beacon in thepresence of target. S:B_(aMB) indicates the increase in fluorescenceresulting from the sensitized emission of the acceptor. S:N_(deg) is theratio of the signal from sensitized emission of the acceptor to thefalse-positive signal.

FIGS. 4 a-4 c show signal-to-noise ratios for dual FRET molecularbeacons with (4 a) a Fam-Cy3 FRET pair, (4 b) a Fam-ROX FRET pair and (4c) a Fam-Texas Red FRET pair. The error bars display the minimum andmaximum ratios calculated for dual FRET molecular beacons separated by3, 4, 5, or 6 bases.

FIG. 5 shows emission spectra for dual FRET molecular beacons with aFam-Texas Red FRET pair. The samples described in the figure wereexcited at a wavelength of 475 nm.

FIG. 6 shows normalized peak emission of the acceptor due to FRET formolecular beacon pairs with a Fam donor and a Cy3, ROX, or Texas Redacceptor. All the intensities were normalized relative to the peakintensity of the Fam-labeled donor beacon bound to target.

FIG. 7 shows the effect of spacing between donor and acceptor beacons onthe fluorescence emission of acceptor dye for dual FRET molecularbeacons with a Fam donor and a Texas Red acceptor. Four differenttargets were tested, separating the donor and acceptor beacons by 3, 4,5, or 6 bases.

FIGS. 8 a and 8 b show time resolved emission spectra obtained in atwo-probe detection assay using Terbium chelate as a donor and (8 a) Cy3as an acceptor and (8 b) ROX as an acceptor. All samples were excited ata wavelength of 325 nm.

FIG. 9 shows time resolved emission spectra generated by a two-probedetection assay utilizing a Europium-labeled oligonucleotide as a donorprobe and a Cy5-labeled oligonucleotide as an acceptor probe.

FIGS. 10 a and 10 b show one-photon (10 a) and two-photon (10 b)excitation spectra of 6-Fam labeled and Cy3 labeled oligonucleotides.

FIGS. 11 a and 11 b shows alternative molecular beacon designs. FIG. 11a shows Conventional molecular beacons have stem sequences that areindependent of the target sequence. FIG. 11 b shows shared-stemmolecular beacons are designed such that one arm of the stemparticipates in both hairpin formation and target hybridization.

FIGS. 12 a and 12 b show examples of the design constraint ofshared-stem molecular beacons with certain stem/probe combinations. FIG.12 a-shows the design of a molecular beacon with a probe length of 19bases and a stem length of 6 bases inadvertently resulted in additionalbases participating in stem formation (circles). FIG. 12 a shows thedesign of a molecular beacon with a probe length of 18 bases and a stemlength of 4 bases inadvertently resulted in an additional baseparticipating in target hybridization (circle).

FIG. 13 shows a comparison of the milting temperature of shared-stem andconventional molecular beacons as determined by the initialconcentrations of probe and target. By fitting the data with a straightline, changes in enthalpy (slope of the fitted line) and entropy(y-intercept) characterizing the phase transition betweenbound-to-target and stem-loop conformations of a molecular beacon wereobtained.

FIG. 14 shows determination of the changes in enthalpy (slope of thefitted line) and entropy (y-intercept) characterizing the phasetransition between bound-to-target and stem-loop conformations forconventional molecular beacons. Similar trend was found for shared-stemmolecular beacons.

FIGS. 15 a and 15 b show determination of the changes in enthalpy (slopeof the fitted line) and entropy (y-intercept) characterizing the phasetransition between bound-to-target and stem-loop conformations for FIG.15 a shared-stem and FIG. 15 b conventional molecular beaconsinteracting with wild-type and mutant targets.

FIG. 16 shows a comparison of melting temperatures as a function of stemlength for conventional and hared-stem molecular beacons in the presenceof wild-type target.

FIGS. 17 a and 17 b show melting behavior of conventional andshared-stem molecular beacons with a 19-base probe and a 5-base stem.FIG. 17 a shows melting curves for conventional and shared-stemmolecular beacons in the presence of wild-type (solid line) and mutant(dashed line) target. FIG. 17 b shows the difference in the fraction ofconventional or shared-stem molecular beacons bound to wild-type andmutant targets.

FIG. 18 shows the difference in the fraction of beacons bound towild-type and mutant targets for shared-stem molecular beacons with stemlengths of 4, 5, and 6 bases. The same trend is true for conventionalmolecular beacons.

FIGS. 19 a and 19 b show a comparison between conventional andshared-stem molecular beacons on FIG. 19 a the on-rate of hybridizationwith target and FIG. 19 b the dissociation constant without target(i.e., the transition between stem-loop hairpin and random-coiledbeacons) for molecular beacons with a 19-base probe length and variousstem lengths.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included therein. Before the presentcompounds, compositions, and methods are disclosed and described, it isto be understood that this invention is not limited to any specificnucleic acid probes, specific nucleic acid targets, specific cell types,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and in the claims, “a” or “an” can mean one or more,depending upon the context in which it is used. Thus, for example,reference to “a nucleic acid probe” can mean that one or more than onenucleic acid probe can be utilized.

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, provides acomposition for the detection of a subject nucleic acid comprising, afirst nucleic acid probe that hybridizes to a first nucleic acid targetsequence on the subject nucleic acid, forms a stem-loop structure whennot bound to the first nucleic acid target sequence, and incorporates aresonance energy transfer donor moiety. This embodiment of the inventionfurther provides a second nucleic acid probe that hybridizes to a secondnucleic acid target sequence on the subject nucleic acid, forms astem-loop structure when not bound to the second nucleic acid targetsequence, and incorporates a resonance energy transfer acceptor moiety.The invention provides that the first nucleic acid target sequence andthe second nucleic acid target sequence are separated by a number ofnucleotides on the subject nucleic acid such that a resonance energytransfer signal from interaction between the donor moiety of the firstnucleic acid probe and the acceptor moiety of the second nucleic acidprobe can be detected to determine hybridization of both the firstnucleic acid probe and the second nucleic acid probe to the subjectnucleic acid. Preferably, the resonance energy transfer signal is aflorescent or luminescent signal.

In an alternative embodiment, the invention provides a first nucleicacid probe that hybridizes to a first nucleic acid target sequence onthe subject nucleic acid, and incorporates a luminescence resonanceenergy transfer lanthanide chelate donor moiety; and second nucleic acidprobe that hybridizes to a second nucleic acid target sequence on thesubject nucleic acid, and incorporates an organic resonance energytransfer acceptor moiety, wherein the first nucleic acid target sequenceand the second nucleic acid target sequence are separated by a number ofnucleotides on the subject nucleic acid such that a luminescenceresonance energy transfer signal from interaction between the lanthanidechelate donor moiety of the first nucleic acid probe and the acceptormoiety of the second nucleic acid probe can be detected to determinehybridization of both the first nucleic acid probe and the secondnucleic acid probe to the subject nucleic acid. In certain embodimentsof this invention, the first nucleic acid probe or second nucleic acidprobe is linear or randomly coiled when not hybridized to the first orsecond nucleic acid target sequences, respectively. In other embodimentsof this invention, the first nucleic acid probe or second nucleic acidprobe forms a stem-loop structure when not hybridized to the first orsecond nucleic acid target sequences, respectively.

In certain preferred embodiments of the invention, the first nucleicacid probe further incorporates a quencher moiety, such that aninteraction between the donor moiety of the first nucleic acid probe andthe quencher moiety can be detected to differentiate between the firstnucleic acid probe in the stem-loop structure and non-stem-loopstructure. Similarly, in other embodiments, the second nucleic acidprobe further incorporates a quencher moiety, such that an interactionbetween the acceptor moiety of the second nucleic acid probe and thequencher moiety can be detected to differentiate between the secondnucleic acid probe in the stem-loop structure and non-stem-loopstructure. In embodiments utilizing a quencher moiety on a nucleic acidprobe, the invention provides that the quencher moiety can be selectedfrom, for example, dabcyl quencher, black hole quencher or Iowa Blackquencher or other moieties well-known in the art to change the resonanceenergy transfer wavelength emission of an unquenched donor or acceptormoiety.

In certain other embodiments, the first nucleic acid probe furtherincorporates a resonance energy transfer moiety pair, such that aresonance energy transfer signal from interaction between the donormoiety and the acceptor moiety on the first nucleic acid probe can bedetected to differentiate between the first nucleic acid probe in thestem-loop structure and non-stem-loop structure. Similarly, otherembodiments provide that the second nucleic acid probe furtherincorporates a resonance energy transfer moiety pair, such that aresonance energy transfer signal from interaction between the donormoiety and the acceptor moiety on the second nucleic acid probe can bedetected to differentiate between the second nucleic acid probe in thestem-loop structure and non-stem-loop structure.

In various embodiments, the first nucleic acid target and the secondnucleic acid target are separated by 1 to 20 nucleotides, or separatedby 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. Asdiscussed below, the preferred number of separating nucleotides willvary depending upon the resonance energy transfer source used, and canbe routinely determined by one of skill in the art in view of thepresent disclosure.

In preferred embodiments, the resonance energy signals are due tofluorescence resonance energy transfer (FRET) or luminescence resonanceenergy transfer (LRET). In embodiments wherein the resonance energytransfer signal is due to fluorescence resonance energy transfer, thedonor moiety can be for example a 6-Fam fluorophore. In embodimentswherein the resonance energy transfer signal is due to fluorescenceresonance energy transfer, the acceptor moieties can be Cy-3, ROX orTexas Red. Additional examples of FRET donor and acceptor moietiesuseful in the present invention are provided below.

In other embodiments, the resonance energy transfer signal is due toluminescence resonance energy transfer (LRET) and the donor moiety is alanthanide chelate. In some preferred embodiments where the resonanceenergy signal is due to LRET, the donor moiety can be Europium orTerbium. Furthermore, in some embodiments where the resonance energysignal is due to LRET, the donor moiety can be a lanthanide chelate suchas DTPA-cytosine, DTPA-cs124, BCPDA, BHHCT, Isocyanato-EDTA, QuantumDye, or W1024 and the acceptor moiety can be Cy-3, ROX or Texas Red. Insome embodiments, due to the range of effective resonance energytransfer of the lanthanide chelate, multiple acceptor moieties may beemployed. The donor moiety can be a lanthanide chelate and the acceptormoiety can be a phycobiliprotein. In certain embodiments, thephycobiliprotein is Red Phycoerythrin (RPE), Blue Phycoerythrin (BPE),or Allophycocyanin (APC). Additional examples of LRET donor and acceptormoieties useful in the present invention are provided below.

In certain embodiments, the invention provides that the first or secondnucleic acid probes each comprise from 5 to 50 nucleotides, 10 to 40nucleotides, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 nucleotides. In other preferred embodiments, the nucleic acidprobes comprise a 2′-O-methyl nucleotide backbone, among manyalternative or synthetic nucleotides, described below. The inventionfurther provides that one end of the first and/or the second nucleicacid probes participates in both stem-loop formation and hybridizationto the target nucleic acid. Such embodiments are referred to herein as ashared-stem molecular beacon, or probe, herein, and are described inmore detail below, particularly in Example 2.

In additional embodiments, the invention provides methods for detectinga subject nucleic acid, comprising combining the composition describedherein with a sample suspected of containing a subject nucleic acid, anddetecting hybridization by differential resonance energy transfer signalto determine the presence or absence, and/or the expression level of thesubject nucleic acid in the sample in vitro or in vivo. In somepreferred embodiments, the methods can be performed in vivo. Therefore,in a preferred embodiment of this method, the sample contains a livingcell. The invention provides that the methods may be performed withsamples comprising living tissues and cells that are taken out of thebody, or that remain in situ.

The methods of the present invention further include detection ofchanges in the levels of expression of a nucleic acid target, or in RNAtranscript, such that alterations of gene expression can be monitored asa result of the dose-dependent cellular response to external stimuli,such as drug molecules, hormones, growth factors, temperature, shearflow, or microgravity, for example. The invention further provides thatthe compositions can be used to visualize, i.e., through fluorescence orluminescence, the location and relative amount of gene expression intissues and cells.

In diagnostic or prognostic detection methods the subject nucleic acidcan comprise a genetic point mutation, deletion, or insertion relativeto a naturally occurring or control nucleic acid. Such screening methodscan permit the detection of the subject nucleic acid indicating thepresence of a genetically associated disease, such as certain cancers,in the sample. There are many well-known examples of genetic mutationsalready in the art that are indicative of a disease state. The methodsinclude the detection of nucleic acids comprising K-ras, survivin, p53,p16, DPC4, or BRCA2. Furthermore, the methods can be used to detect theamount of a subject nucleic acid being produced by an organism forpurposes other than diagnosis or prognosis of a disease or condition.Resonance energy transfer detections of the present invention can beperformed with the assistance of single- or multiple-photon microscopy,time-resolved fluorescence microscopy or fluorescence endoscopy, asdetailed below.

The invention further provides kits for the detection of a subjectnucleic acid comprising the nucleic acid probe compositions describedherein, necessary reagents and instructions for practicing the methodsof detection. Such alternative compositions, methods and kits thereforare described in more detail by way of the examples, and still otherswill be apparent to one of skill in the art in view of the presentdisclosure.

One embodiment of the present invention provides compositions andmethods that measure a resonance energy transfer, for example, afluorescent signal due to FRET or LRET as a result of direct interactionbetween two molecular beacons when hybridized to the same target nucleicacid of interest. This method can dramatically reduce false-positivesignals in gene detection and quantification in living cells. As shownin FIG. 1 and alternatively in FIG. 11, this approach utilizes a pair ofmolecular beacons, one with a donor fluorophore and a second with anacceptor fluorophore. Probe sequences are chosen such that the molecularbeacons hybridize adjacent to each other on a single nucleic acid targetin a way that positions their respective fluorophores in optimalconfiguration for FRET (Mergny et al., 1994; Sixou et al. 1994).Emission from the acceptor fluorophore serves as a positive signal inthe FRET based detection assay.

If acceptor and donor fluorophores are well matched, excitation of thedonor can be achieved at a wavelength that has little or no capacity toexcite the acceptor; excitation of the acceptor will therefore onlyoccur if both molecular beacons are hybridized to the same targetnucleic acid and FRET occurs. Molecular beacons that are degraded oropen due to protein interactions will result in the presence ofunquenched fluorophore, however, fluorescence emitted from these speciesis different in character from the signal obtained from donor/acceptorFRET pair, making background and true positive signal more readilydifferentiated. Thus, by detecting FRET instead of directsingle-molecule fluorescence, nucleic acid probe/target binding eventscan be distinguished from false-positives.

In contrast to prior labeling of two linear oligonucleotide probes withdonor and acceptor fluorophores, the stem-loop hairpin structure ofmolecular beacons offers further reduction in background fluorescence aswell as enhanced specificity, which is helpful particularly whendetection of allelic variants or point mutations is desired (Bonnet etal., 1999; Tsourkas et al., 2002a).

Further benefit from another embodiment of the dual energy transfermolecular beacons and method of the present invention can be achieved byemploying an oligonucleotide probe with a lanthanide chelate as thedonor and a molecular beacon with a traditional organic fluorophore asthe acceptor (reporter) moiety. In contrast to organic fluorophores thathave a fluorescence lifetime of ˜10 ns, lanthanide chelates can haveemission lifetimes greater than 1 ms (Sueda et al; 2000; Evangelista etal., 1988). The mechanism that is responsible for the long lifetimeemission of lanthanide chelates is complex and involves energy transferfrom the triplet state of the aromatic ligand. Specifically, uponexcitation the ligand is excited to its singlet state and then undergoesan intersystem transition to its triplet state, whereas the energy iseither quenched by water molecules or transferred to the lanthanide ion.Fluorescence is then emitted from the lanthanide ion as it returns tothe ground state (Lemmetyinen et al., 2000). Since such fluorescenceemission does not result from a singlet-to-singlet transition, the useof lanthanide chelates as a donor results in luminescent resonanceenergy transfer (LRET). Therefore, by using pulse excitation andtime-gating techniques, it is possible to selectively record emissionafter the background fluorescence from organic dyes, scattering, andautofluorescence has decayed (Yuan et al, 1998; Lopez et al, 1993). Theonly signals remaining in this long-time domain are the emission fromthe lanthanide chelate and from acceptor fluorophores that haveparticipated in LRET. In this case the narrow emission peaks of alanthanide chelate render the background fluorescence close to zero atcertain wavelengths, leading to extremely large signal-to-backgroundratio. The donor probe in a LRET pair can be a simple linear probe,i.e., neither quencher nor hairpin structure are necessary.

Furthermore, the invention provides a design variant for molecularbeacons where one arm of the stem participates in both hairpin formationand target hybridization, referred to herein as “shared-stem” molecularbeacons. In contrast, conventional molecular beacons are designed suchthat the loop sequence is complementary to the target while the stemsequences are self-complementary but unrelated to the target sequence.This new design offers certain advantages over conventional molecularbeacon design, especially in two-probe fluorescence resonance energytransfer (FRET) assays (Cardullo et al., 1998; Sei-Iida et al., 2000;Tsuji et al., 2000; Tsuji et al., 2001; Tsourkas and Bao 2001). Theinvention provides the thermodynamic and kinetic properties of bothshared-stem and conventional molecular beacons and makes a systematiccomparison between them. In particular, the present descriptionquantifies the changes in enthalpy and entropy upon the formation ofprobe/target duplexes as determined by the probe and stem lengths.Further provided herein is a study of the melting behavior, specificity,and hybridization on-rate depend on the stem length of molecularbeacons, such that one of skill in the art may make and use a variety ofembodiments to suit the specific purposes of each situation.

The nucleic acid probes of the invention utilize the principle ofresonance energy transfer between a donor moiety and an acceptor moiety.In a preferred embodiment, the resonance energy transfer is fluorescenceresonance energy transfer (FRET), in which the first and second probesare labeled with donor and acceptor moieties, respectively, wherein thedonor moiety is a fluorophore and the acceptor moiety may be afluorophore, such that fluorescent energy emitted by the donor moiety isabsorbed by the acceptor moiety when both probes are hybridized to thefirst and second target sequences respectively on the same nucleic acidsubject. In one embodiment of the present invention, the acceptor moietyis a fluorophore that releases the energy absorbed from the donor at adifferent wavelength; the emissions of the acceptor may then be measuredto assess the progress of the hybridization reaction.

In a preferred embodiment, the probe is a hairpin stem-loop structure(often referred to in the art as a molecular beacon) that containseither a donor or acceptor moiety and optionally a quencher moiety, suchthat the quencher moiety reduces the fluorescence of the donor oracceptor when the probe is in the stem-loop structure (i.e., nothybridized). When the probe is hybridized to the target nucleic acid inthis embodiment, its conformation changes, eliminating the quenchingeffect, and the resulting fluorescence of the donor or acceptor moietymay be detected.

In an alternative embodiment, the present invention provides a nucleicacid probe that forms a hairpin stem-loop structure in which resonanceenergy transfer will decrease when the probe is hybridized with thetarget nucleic acid. In such an embodiment, the quencher moiety on thefirst probe is replaced with a reciprocating moiety to form a resonanceenergy transfer moiety pair, and the differential in resonance energytransfer is detectable between the hairpin stem-loop structure and anon-stem-loop structure. Alternatively, the quencher moiety on thesecond probe is replaced with a reciprocating moiety to form a resonanceenergy transfer moiety pair, and the differential in resonance energytransfer is detectable between the hairpin stem-loop structure and anon-stem-loop structure. In such embodiments of the present invention, athird resonance energy transfer moiety pair forms by the dual probes, adonor moiety on the first probe, and an acceptor moiety on the secondprobe, such that the resonance energy transfer signal due to theinteraction of donor and acceptor may be measured to assess the progressof the hybridization reaction of both probes on the subject nucleicacid.

In another embodiment, the present invention provides that one of thenucleic acid probes is linear (non-stem-loop) and the probes areseparately labeled with lanthanide chelator donor and organic acceptormoieties, such that resonance energy transfer will occur when thenucleic acid probes are hybridized. In yet another embodiment, theinvention uses a pair of such linear primers, one labeled with alanthanide donor and another with an organic acceptor moiety,respectively.

One aspect of the invention pertains to nucleic acids sufficient for useas hybridization probes for the identification of a target nucleic acid(e.g., DNA or mRNA). As used herein, the term “nucleic acid” is intendedto include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. As referred to herein, nucleic acids that are “complementary”can be perfectly or imperfectly complementary, as long as the desiredproperty resulting from the complementarity is not lost, e.g., abilityto hybridize.

The nucleic acids of the present invention may be substantially isolatedor alternatively unpurified. An “isolated” or “purified” nucleic acid isone that is substantially separated from other nucleic acid moleculesthat are present in the natural source of the nucleic acid. Preferably,an “isolated” nucleic acid is substantially free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. Moreover, an “isolated” nucleic acidmolecule can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. (see,Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

The probe typically comprises substantially purified nucleic acid. Thenucleic acid probe typically comprises a region of nucleotide sequencethat hybridizes to at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45or 50 consecutive nucleotides of a target nucleic acid. The targetnucleic acid can be a sense strand of one of the target nucleic acidsequences, an anti-sense sequence, or naturally occurring mutantsthereof. Preferably, the nucleic acid target is an mRNA.

Probes based on the nucleotide sequences can be used to detect oramplify transcripts or genomic sequences encoding the same or homologousproteins. In other embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, anenzyme, or an enzyme co-factor. Such probes can be used as a part of agenomic marker test kit for identifying cells which express a particularprotein, such as by measuring a level of the protein-encoding nucleicacid in a sample of cells, e.g., detecting the target nucleic acid mRNAlevels or determining whether the gene encoding the mRNA has beenmutated or deleted.

In an additional preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a nucleic acid probe sequence thathybridizes, e.g., hybridizes under stringent conditions, to a targetnucleotide sequence of interest. These hybridization conditions includewashing with a solution having a salt concentration of about 0.02 molarat pH 7 at about 60° C. As used herein, the term “hybridizes understringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%homologous to each other typically remain hybridized to each other.Preferably, the conditions are such that sequences at least about 65%,more preferably at least about 70%, and even more preferably at leastabout 75% or more homologous to each other typically remain hybridizedto each other. Such stringent conditions are known to those skilled inthe art and can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989) 6.3.1-6.3.6. A preferred, non-limiting exampleof stringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

The nucleic acid probes of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, so long as it isstill capable of hybridizing to the desired target nucleic acid. Inaddition to being labeled with a resonance energy transfer moiety, thenucleic acid sequence can be modified at the base moiety, sugar moiety,or phosphate backbone, and may include other appending groups or labels,so long as it is still capable of priming the desired amplificationreaction, or functioning as a blocking oligonucleotide, as the case maybe.

For example, a nucleic acid probe of the present invention can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the complimentary nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. A preferred example of a class of modified nucleotides which canbe used to generate the nucleic acid probes is a 2′-O-methyl nucleotide.Additional examples of modified nucleotides which can be used togenerate the nucleic acid probes include for example 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-

5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil,(acp3)w, and 2,6-diaminopurine.

In another embodiment, the nucleic acid probe of the present inventioncomprises at least one modified sugar moiety selected from the groupincluding but not limited to arabinose, 2-fluoroarabinose, xylulose, andhexose. In yet another embodiment, the nucleic acid probe of the presentinvention comprises at least one modified phosphate backbone selectedfrom the group consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof. As stated above, a preferred example of a modified nucleotidewhich can be used to generate the nucleic acid probes is a 2′-O-methylnucleotide.

Nucleic acid probes of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

Once the desired oligonucleotide is synthesized, it is cleaved from thesolid support on which it was synthesized and treated, by methods knownin the art, to remove any protecting groups present. The oligonucleotidemay then be purified by any method known in the art, includingextraction and gel purification. The concentration and purity of theoligonucleotide may be determined by examining oligonucleotide that hasbeen separated on an acrylamide gel, or by measuring the optical densityat 260 nm in a spectrophotometer.

Nucleic acid probes of the invention may be labeled with donor andacceptor moieties during chemical synthesis or the label may be attachedafter synthesis by methods known in the art. In a specific embodiment,the following donor and acceptor pairs are used: a luminescentlanthanide chelate, e.g., terbium chelate or lanthanide chelate, is usedas the donor, and an organic dye such as fluorescein, rhodamine or CY-5,is used as the acceptor. Preferably, terbium is used as a donor andfluorescein or rhodamine as an acceptor, or europium is used as a donorand CY-5 as an acceptor. In another specific embodiment, the donor isfluorescent, e.g. fluorescein, rhodamine or CY-5, and the acceptor isluminescent, e.g. a lanthanide chelate. In yet another embodiment, theenergy donor is luminescent, e.g., a lanthanide chelate, and the energyacceptor may be non-fluorescent.

In another specific embodiment, the donor moiety is a fluorophore. Inanother specific embodiment, both donor and acceptor moieties arefluorophores. Suitable moieties that can be selected as donor oracceptors in FRET pairs are set below:

4-acetamido-4′-isothiocyanatostilbene-2,2′Disulfonic Acid Acridine andDerivatives:

-   -   acridine    -   acridine isothiocyanate    -   5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)    -   4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate        (LuciferYellow VS)    -   N-(4-anilino-1-naphthyl)maleimide    -   Anthranilamide    -   Brilliant Yellow        coumarin and derivatives:    -   coumarin    -   7-amino-4-methylcoumarin (AMC, Coumarin 120)    -   7-amino-4-trifluoromethylcoumarin (Coumarin 151)    -   cyanosine    -   4′-6-diaminidino-2-phenylindole (DAPI)    -   5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red)    -   7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin    -   diethylenetriamine pentaacetate    -   4-(4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid    -   4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid    -   5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl        chloride)    -   4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)    -   4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC)        eosin and derivatives:    -   eosin    -   eosin isothiocyanate        erythrosin and derivatives:    -   erythrosin B    -   erythrosin isothiocyanate    -   ethidium        fluorescein and derivatives:    -   5-carboxyfluorescein (FAM)    -   5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)    -   2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE)    -   fluorescein    -   fluorescein isothiocyanate    -   QFITC (XRITC)    -   fluorescamine    -   IR144    -   IR1446    -   Malachite Green isothiocyanate    -   4-methylumbelliferone    -   ortho cresolphthalein    -   nitrotyrosine    -   pararosaniline    -   Phenol Red    -   B-phycoerythrin    -   o-phthaldialdehyde        pyrene and derivatives:    -   pyrene    -   pyrene butyrate    -   succinimidyl 1-pyrene butyrate    -   Reactive Red 4 (Cibacron® Brilliant Red 3B-A)        rhodamine and derivatives:    -   6-carboxy-X-rhodamine (ROX)    -   6-carboxyrhodamine (R6G)    -   lissamine rhodamine B sulfonyl chloride    -   rhodamine (Rhod)    -   rhodamine B    -   rhodamine 123    -   rhodamine X isothiocyanate    -   sulforhodamine B    -   sulforhodamine 101    -   sulfonyl chloride derivative of sulforhodamine 101 (Texas Red)    -   N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA)    -   tetramethyl rhodamine    -   tetramethyl rhodamine isothiocyanate (TRITC)    -   riboflavin    -   rosolic acid    -   terbium chelate derivatives

One of ordinary skill in the art can easily determine, using art-knowntechniques of spectrophotometry, which fluorophores will make suitabledonor-acceptor FRET pairs. For example, FAM (which has an emissionmaximum of 525 nm) is a suitable donor for TAMRA, ROX, and R6G (all ofwhich have an excitation maximum of 514 nm) in a FRET pair. Probes arepreferably modified during synthesis, such that a modified T-base isintroduced into a designated position by the use of Amino-Modifier C6 dT(Glen Research), and a primary amino group is incorporated on themodified T-base, as described by Ju et al. Proc. Natl. Acad. Sci. USA92:4347-4351). These modifications may be used for subsequentincorporation of fluorescent dyes into designated positions of thenucleic acid probes of the present invention.

The optimal distance between the donor and acceptor moieties will bethat distance wherein the emissions of the donor moiety are maximallyabsorbed by the acceptor moiety. This optimal distance varies with thespecific moieties used, and may be easily determined by one of ordinaryskill in the art using well-known techniques. For energy transfer inwhich it is desired that the acceptor moiety be a fluorophore that emitsenergy to be detected, the donor and acceptor fluorophores arepreferably separated when hybridized to target nucleic acid by adistance of up to 30 nucleotides, more preferably from 1-20 nucleotides,and still more preferably from 2 to 10 nucleotides and more preferablyseparated by 3, 4, 5, 6, 7, 8 and 9 nucleotides. For energy transferwherein it is desired that the acceptor moiety quench the emissions ofthe donor, the donor and acceptor moieties are preferably separated by adistance of less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide (e.g.,on the opposite strand, complementary nucleotides of a duplexstructure), although a 5 nucleotide distance (one helical turn) is alsoadvantageous for use.

In yet another embodiment, the nucleic acid probes of the invention maybe further labeled with any other art-known detectable marker, includingradioactive labels such as ³²P, ³⁵S, ³H, and the like, or with enzymaticmarkers that produce detectable signals when a particular chemicalreaction is conducted, such as alkaline phosphatase or horseradishperoxidase. Such enzymatic markers are preferably heat stable, so as tosurvive the denaturing steps of the amplification process. Nucleic acidprobes may also be indirectly labeled by incorporating a nucleotidelinked covalently to a hapten or to a molecule such as biotin, to whicha labeled avidin molecule may be bound, or digoxygenin, to which alabeled anti-digoxygenin antibody may be bound. Nucleic acid probes maybe supplementally labeled during chemical synthesis or the supplementallabel may be attached after synthesis by methods known in the art.

The nucleic acid probes of the invention have use in nucleic aciddetection, or amplification reactions as primers, or in the case oftriamplification, blocking oligonucleotides, to detect or measure anucleic acid product of the amplification, thereby detecting ormeasuring a target nucleic acid in a sample that is complementary to a3′ primer sequence. Accordingly, the nucleic acid probes of theinvention can be used in methods of diagnosis, wherein a sequence iscomplementary to a sequence (e.g., genomic) of an infectious diseaseagent, e.g. of human disease including but not limited to viruses,bacteria, parasites, and fungi, thereby diagnosing the presence of theinfectious agent in a sample of nucleic acid from a patient. The targetnucleic acid can be genomic or cDNA or mRNA or synthetic, human oranimal, or of a microorganism, etc.

In one embodiment the inventor provides a useful screening tool for drugdiscovery where a rapid specific and sensitive assay can detect in vivochanges in the expansion role of protein transcripts of interest, eitherat a steady state or in response to the administration of drugcandidates. In another embodiment that can be used in the diagnosis orprognosis of a disease or disorder, the target sequence is a naturallyoccurring or wild type human genomic or RNA or cDNA sequence, mutationof which is implicated in the presence of a human disease or disorder,or alternatively, the target sequence can be the mutated sequence. Insuch an embodiment, optionally, the amplification reaction can berepeated for the same sample with different sets of probes that amplify,respectively, the naturally occurring sequence or the mutated version.By way of example, the mutation can be an insertion, substitution,and/or deletion of one or more nucleotides, or a translocation.

EXAMPLES Example 1

Dual Nucleic Acid Probes

Oligonucleotide Synthesis. Oligonucleotide probes and targets weresynthesized using standard phosphoramidite chemistry on an AppliedBiosystems model 394 automated DNA synthesizer (Foster City, Calif.).Molecular beacons were purified using dual reverse phase (RP) plusion-exchange (IE) high performance liquid chromatography (HPLC) on aWaters Model 600E HPLC system (Millipore Corp., Milford, Mass.). ForRP-HPLC purification, oligonucleotides were loaded on a Hamilton PRP-1column and eluted with a linear 5% to 50% acetonitrile gradient in 0.1 Mtriethyl-ammonium acetate (TEAA) pH 7.2 over 40 minutes. Theoligonucleotides were additionally purified by IE-HPLC using a Source™column (Amersham Pharmacia Biotech, Piscataway, N.J.) and eluted with alinear 0% to 50% 1 M LiCl gradient in 0.1 M Tris pH 8.0 over 40 minutes.Unmodified (target) oligonucleotides were purified using polyacrylamidegel electrophoresis. All oligonucleotides were synthesized at IntegratedDNA Technologies, Inc. (Coralville, Iowa).

Probe and Target Design. All oligonucleotide probes were designed to becomplementary in antisense orientation to the human GAPDH gene, asillustrated in FIG. 2. Specifically, a dabcyl quencher was attached tothe 5′-end and a 6-Fam fluorophore was attached to the 3′-end of donormolecular beacons; a dabcyl quencher was attached to the 3′-end andeither a Cyanine 3 (Cy3), 6-carboxyrhodamine (ROX), or Texas Redfluorophore was attached to the 5′-end of acceptor molecular beacons.The stem sequence was designed to participate in both hairpin formationand target hybridization (Tsourkas et al., 2002b). This beacon designwas chosen to help fix the relative distance between the donor andacceptor fluorophores and improve energy transfer efficiency. Both thedonor and acceptor beacons were designed with a probe length of 18 basesand a stem length of 5 bases. The probe length is defined as the portionof the molecular beacon that is complementary to the target. Thesynthetic wild-type GAPDH target has 4-base gap between the donor dyeand the acceptor dye. Gap spacing was adjusted to 3, 5, and 6 bases byeither removing a guanine residue or adding 1 or 2 thymine residues, asshown in Table 1.

TABLE 1 Design of Probes and Target Oligonucleotides Name Sequence(5′-3′) Note Fam donor-MB⁽¹⁾ Dabcyl-ccacaTGATGGCATGGACTGTGG-Fam Probe18/Stem 5 SEQ ID NO:1 Tb donor probe TGATGGCATGGACTGTGG-DTPA-cs124-(Tb)Probe 18/Stem 0 SEQ ID NO:2 Cy3 acceptor-MBCy3-GAGTCCTTCCACGATACCgactc-Dabcyl Probe 18/Stem 5 SEQ ID NO:3 ROXacceptor-MB ROX-GAGTCCTTCCACGATACCgactc-Dabcyl Probe 18/Stem 5 SEQ IDNO:4 Texas Red Texas Red-GAGTCCTTCCACGATACCgactc-Dabcyl Probe 18/Stem 5acceptor-MB SEQ ID NO:5 Target^((2) n-1)ACTTTGGTATCGTGGAAGGACTCATACCACAGTCCATGCCATCACTGCC 3 base gap SEQ ID NO:6Target n (WT) ACTTTGGTATCGTGGAAGGACTCATGACCACAGTCCATGCCATCACTGCC 4 basegap SEQ ID NO:7 Target n+1ACTTTGGTATCGTGGAAGGACTCATTGACCACAGTCCATGCCATCACTGCC 5 base gap SEQ IDNO:8 Target n+2 ACTTTGGTATCGTGGAAGGACTCATTTGACCACAGTCCATGCCATCACTGCC 6base gap SEQ ID NO:9 ⁽¹⁾MB = Molecular Beacon. Lower case = bases addedto create stem domains. Upper case = probe-target hybridizing domains.Upper case bold = bases participating in both stem formation and targetbinding ⁽²⁾Underscore = 18 base sequence complementary to MB targetbinding domains. n = 4 bases the wild-type gap size

Lanthanide Chelate Synthesis. A linear oligonucleotide with a probelength of 18 bases was labeled at its 3′-end with adiethylenetriaminepentaacetic acid (DTPA) chelate covalently joined to asensitizer, cs124 (Cooper and Sammes, 2000). As demonstrated in Table 1,the sequence of this linear probe was identical to the probe domain ofthe donor molecular beacons specific for exon 7 of the human GAPDH gene.

The lanthanide chelate was prepared by first dissolving DTPA (500 mg,1.4 μmole) in 30 mL of DMF and 1 mL of triethylamine. Cs124 (240 mg, 1.4μmole), dissolved in 4 mL of DMF, was then added dropwise and mixed for30 minutes. To this mixture, 5 mL (75 μmole) of ethylenediamine (EDA)was added and stirred at room temperature for two hours. The mixture wasthen stored in the refrigerator overnight. A slightly off-whiteprecipitate had formed and was centrifuged down further. The DMFsupernatant was removed and the pellet was washed with isopropanolseveral times and then with ether resulting in a fine white powder,which was dried under a vacuum for 2 hours. The powder was resuspendedin water and RP-HPLC purified using a Hamilton PRP-1 column. The samplewas eluted with a linear 0% to 30% acetonitrile gradient in 0.1 M TEAApH 7.2 over 20 minutes at a flow rate of 10 mL/min. The first peak wascollected, and the DTPA-cs124 product was dried and reconstituted to aconcentration of 15 mM in 0.1 M Borate Buffer, pH 8.5.

Disuccinimidyl suberate (1.84 mg, 5 μmoles; Pierce Chemical) wasdissolved in 100 μL of DMSO and added to 0.1 μmoles of oligonucleotideswith a 3′-amine, dissolved in 100 uL of DMSO. The mixture was incubatedat 40° C. for 2 hours. The oligonucleotides were then acetoneprecipitated and reconstituted in 100 μl of 0.1 M sodium borate pH 8.5.50 uL of 15 mM DTPA-cs124-EDA product in borate buffer was added to theoligonucleotide solution and mixed overnight.Oligonucleotide-DTPA-cs124-EDA conjugates were purified usingreversed-phase (RP) HPLC. The oligonucleotides were loaded on a PRP-1column and eluted with a linear 5% to 50% acetonitrile gradient in 0.1 MTEAA pH 7.2 over 40 minutes. The collected peak was lyophilized andreconstituted in dH₂O at 5 μM. TbCl₃ (Terbium) dissolved in PBS was thenadded to the sample at a 10:1 molar ratio and incubated at roomtemperature for 30 minutes. The Europium chelates were synthesizedfollowing the same protocol.

Hybridization and Detection Assays. Hybridization experiments wereconducted with 50 pmoles of donor beacon, 50 pmoles of acceptor beaconand 50 pmoles of complementary target in a total volume of 100 μL (0.5μM). All experiments were conducted at 37° C. in HB buffer containing 10mM KCl, 5 mM MgCl₂, and 10 mM Tris-HCl, pH 7.5, which was supplementedwith 1% Bovine Albumin Serum to block non-specific interactions with themicroplate. The samples were mixed and allowed to equilibrate at 37° C.for 20 minutes before performing fluorometry. A Safire microplatefluorometer (Tecan, Zurich, Switzerland) was used to excite the donorbeacons and detect resulting emission (500 nm to 650 μm) in FRETmeasurements. The excitation wavelength was varied from 395 nm to 495 nmto determine the wavelength that resulted in the maximal FRET betweenthe donor and acceptor molecules. In a two-photon experimental set-up,the excitation spectra of Fam- and Cy3-labeled linear oligonucleotideswere obtained. A tunable laser was adjusted to excite the samples atwavelengths ranging from 700 nm to 875 nm. The fluorescence emissionbetween 505 nm and 555 nm was detected from the Fam sample and theemission between 590 and 650 nm was detected from the Cy3 sample usingultra-sensitive, low noise avalanche photodiodes.

For LRET measurements, the Terbium and Europium donor probes wereexcited at a wavelength of 325 nm, and the emission was recorded from500 nm to 650 nm for assays with Terbium donors, and from 550 nm to 750nm for assays involving Europium donors. The emission detection had alag time of 50 μs with an integration time of 1 ms. The maximalexcitation and emission wavelengths of the organic and lanthanide dyesused in this study are summarized in Table 2.

TABLE 2 Maximal Excitation and Emission Wavelengths of Organic andLanthanide Dyes Extinction Excitation Emission Coefficient Dye Molecule(nm) (nm) (M⁻¹ cm⁻¹) Note 6-Fam 494 518 83,000 Donor Terbium Chelate300-340 546 10,000-35,000 Donor Europium Chelate 300-340 62010,000-35,000 Donor Cy3 552 570 150,000  Acceptor Rox 585 605 82,000Acceptor Taxes Red 583 603 116,000  Acceptor

FRET of Organic Dye Pairs. A series of solution-phase assays wereconducted to determine whether the signal generated by a pair ofmolecular beacons hybridized to the same target oligonucleotide can bedifferentiated from the signal due to false-positive events. For organicdye pairs, the same donor beacon (i.e., a molecular beacon with afluorescent donor dye) was tested with three acceptor (reporter) beaconsfor the magnitudes of background signal and positive (FRET) signal.Here, “background” is defined as fluorescence detected from one or bothbeacons in the absence of target or from either beacon alone in thepresence of target. Thus background represents any fluorescence emissiondetected in the absence of a FRET event due to the simultaneoushybridization of the donor and acceptor beacons to the same target. Iffluorescence excitation is limited to wavelengths λ_(e) optimal for thedonor fluorophore and signal detection is limited to wavelengths λ_(d)optimal for the acceptor (reporter) fluorophore, fluorescent signalshould be low unless both beacons hybridize to the same target and FREToccurs. However, since fluorescence from organic fluorophores occursover a broad range of wavelengths, it is possible for fluorescenceemission from the donor at λ_(d) and from the acceptor due to directexcitation at λ_(e) to contribute to background. “Positive signal” isdefined as FRET-induced fluorescence detected when both beacons arebound to the same target, again restricting excitation to wavelengthsλ_(e) and limiting detection to wavelengths λ_(d).

As illustrated in Table 1, the donor molecular beacon was labeled with6-Fam on the 3′-end, and the acceptor molecular beacons were labeledwith Cy3, ROX, or Texas Red on the 5′-end. Sequences of donor andacceptor molecular beacons were chosen to be complementary to adjacentsites within exon 6 and exon 7 of the human GAPDH gene and werepositioned with a four base separation between donor and acceptorfluorophores when wild-type target was used. Four types of assays wereperformed with each donor/acceptor beacon pair: (1) both donor andacceptor beacons in the absence of target, with a typical emissionspectrum (i.e., fluorescence intensity as a function of wavelength)shown as curve a in FIG. 3 (spectrum a); (2) donor beacon only in thepresence of target, with a typical emission spectrum shown as curve b inFIG. 3 (spectrum b); (3) acceptor beacon only in the presence of target,with a typical emission spectrum shown as curve c in FIG. 3 (spectrumc); and (4) both donor and acceptor beacons in the presence of target,with a typical emission spectrum shown as curve d in FIG. 3 (spectrumd). Assays (2) and (3) simulate the limiting false positive scenariowhere most of the molecular beacons open as a result of nucleasedegradation, denaturation, or non-specific protein interactions (whichhereafter will collectively be referred to as ‘degraded’ beacons).

To illustrate the advantages of using duel FRET molecular beacons and tocompare the performance of different acceptor molecular beacons, severalsignal-to-background ratios were calculated. As illustrated in FIG. 3,the first is S:B_(dMB), the ratio of the peak fluorescence intensity ofemission spectrum b defined above for donor molecular beacons (dMB) tothat of emission spectrum a at the same wavelength. Although spectrum awas generated with both the donor and acceptor beacons in solution, thefluorescence signal is largely due to the donor beacons, for theemission of the acceptor beacons at the corresponding wavelength isalmost zero, as can be seen from curve c in FIG. 3. Thus, S:B_(dMB)represents the signal-to-background ratio of the conventional singlemolecular beacon assay. The second is S:B_(aMB), the ratio of the peakfluorescence intensity of emission spectrum d of the acceptor molecularbeacon (aMB) due to FRET to that of emission spectrum a at the samewavelength. Clearly, S:B_(aMB) represents the signal-to-background ratioof the dual FRET molecular beacons assay without degraded beacons. Thethird one, S:B_(deg), is defined as the ratio of the peak fluorescenceintensity of emission spectrum d due to FRET to that of emissionspectrum b or c at the same wavelength, whichever is higher. S:B_(deg)represents the signal-to-background ratio of the dual FRET molecularbeacons assay for the limiting case that most of the donor and acceptorbeacons are being degraded. Here we assume that, with up to 1×10⁵molecular beacons per cell, the probability of having both degradeddonor and acceptor beacons at the same spatial location (i.e., within acylinder of 0.2 μm in diameter and 1 μm in thickness) in a fluorescenceimaging assay is small. This is especially true considering that, withchemical modifications of the beacon backbone, only a small fraction(<50%) of the molecular beacons would be degraded in an intracellularenvironment. It is worth mentioning that all the signal-to-backgroundratios discussed above change with the donor excitation wavelength.

As shown in FIG. 4 a, S:B_(aMB) of the FRET assay with Cy3-labeledacceptor beacons was almost identical to S:B_(dMB) of the donor beaconalone over the entire range of excitation wavelengths tested. Neitherparameter varied significantly, ranging between 20 and 25, as theexcitation wavelength λ was increased. The dual FRET molecular beacons,however, did generate a signal 2 to 3 times stronger compared with thatof degraded beacons, i.e., S:B_(deg) has a value of 2-3, whileconventional molecular beacons cannot differentiate between signals dueto degraded and hybridized probes. Signal enhancement upon molecularbeacon/target binding is strongly affected by cation concentration andtemperature. In this case, the S:B_(deg) was relatively low since theassay temperature of 37° C. was near the stem melting temperature.

When a ROX fluorophore was used as the acceptor dye, S:B_(aMB) was foundto be ˜10, about half of S:B_(dMB), at low excitation wavelengths λ(e.g., 395 nm to 425 nm), as shown in FIG. 4 b. However, when λ wasincreased, S:B_(aMB) also increased. In fact, when λ became larger than460 nm, S:B_(aMB) was higher than S:B_(dMB), reaching values above 30.This indicates that the dual FRET molecular beacons with a Fam-ROX FRETpair can perform better than the conventional molecular beacons even inthe absence of beacon degradation issues. The value of S:B_(deg) alsoincreased with increasing λ, reaching values close to 5 at λ=455 nm,remaining between 4 and 5 for wavelengths ranging from 455 nm to 495 nm.

Acceptor beacons labeled with Texas Red were found to perform the bestamong the three acceptor dyes considered. Value of S:B_(aMB) increasedfrom 10 to nearly 50 as λ was increased from 395 nm to 495 nm. Moreover,as λ was increased from 455 nm to 475 nm the value of S:B_(deg)increased from ˜2 to over 10 and remained around 10 for λ>475 nm, asdemonstrated in FIG. 4 c. Therefore, the signal generated by binding ofboth donor and acceptor beacons to a target could be 10 times brighterthan false-positive signals. An example of the spectra generated usingthe Fam-Texas Red FRET pair is given in FIG. 5.

Although the performance of dual FRET molecular beacons is better thannon-FRET molecular beacons due to increased signal-to-background ratioand the ability to differentiate between bound and degraded molecularbeacons, the peak fluorescence intensity of the acceptor beacons wastypically lower than that of the Fam-labeled donor beacons, as shown inFIG. 6. Specifically, at wavelengths where optimal FRETsignal-to-background ratios were obtained, for Fam-Texas Red FRET pairthe peak signal intensity of the acceptor was only about 40% of thatemitted by the Fam-donor alone, and only about 25% for the Fam-ROX FRETpair, which may limit ultimate sensitivity.

The efficiency E of fluorescence resonance energy transfer between theadonor and acceptor fluorophores varies according toE=1/(1+R ⁶ /R ₀ ⁶)  (1)where R is the distance between donor and acceptor dyes, and R₀ is theFörster energy transfer distance or the distance at which E=0.5. Fortypical fluorophores R₀=1˜5 nm. Equation (1) implies that the gap (i.e.,the number of bases) between the donor and acceptor beacons should bekept small. However, too small a gap size may result in stericinterference between fluorophores or might lead to other interactionbetween donor and acceptor (such as ground state quenching), which isunfavorable. The gap size can influence the relative orientation of thefluorophores, also affecting energy transfer efficiency. A gap size of 8bases was found to be optimal for energy transfer in the single-strandedrandom-coil conformation (Ju et al., 1995; Hung et al., 1997). Further,base composition can influence fluorescence efficiency. The combinationof a fluorescein dye attached to a guanine base can decrease peakfluorescence intensity by as much as 30% (M. Behlke, unpublishedobservation). To optimize design parameters, hybridization experimentswere conducted using targets that separate the donor and acceptorbeacons by 3, 4, 5, and 6 bases. The nucleotides closest to theprobe-binding region were identical for each target sequences. When thedistance between the donor and acceptor beacons was increased from 3 to6 bases, there was a slight increase in the FRET signal intensity, asdemonstrated by the curves displayed in FIG. 7. This trend was found tobe the same for all the acceptor fluorophores studied.

LRET of Lanthanide Dyes. Conventional organic dyes used for FRET assaysare limited by problems associated with the overlapping ofdonor/acceptor excitation and emission spectra. To dramatically improvethe signal-to-background ratio, the inventor takes advantage of thesharp emission peaks and the long lifetime of a lanthanide chelate (Liand Selvin, 1997; Cooper and Sammes, 2000). Specifically, a lanthanidedonor is substituted for the Fam donor and modified the detection systemto employ time-resolved spectroscopy. The lanthanide donor was a linearoligonucleotide probe labeled at its 3′-end with the Terbium chelateDTPA-cs124 (Table 1). The same series of acceptor molecular beacons weretested as before, including beacons with Cy3, ROX, or Texas Redfluorophores. Note that the use of lanthanide donor allows for shorterwavelength excitation, as demonstrated in Table 2.

The results of LRET experiments with a lanthanide donor are shown inFIGS. 8 a, b. As shown by curve (1) in FIG. 8 a, at 325 nm excitation,when the Terbium-chelate labeled donor probes bound to Cy3 labeledacceptor beacons, they exhibited several sharp emission peaks separatedby valleys with fluorescence intensity close to zero; while thefluorescence emission from acceptor molecular beacons alone hybridizedto targets was extremely low, as shown by curve (2). With binding ofboth donor probes with Terbium-chelate and acceptor molecular beacons totarget, a sensitized emission of the acceptor due to LRET was observed,shown as curve (3). As clearly demonstrated by the insert in FIG. 8 a,at emission wavelengths where background from the lanthanide donor wasnear zero, extremely high signal-to-background ratios were observed. ForCy3-labeled acceptor beacon, the optimal detection wavelength is around573 nm. Similar features were exhibited in FIG. 8 b in which the timeresolved emission spectra obtained with 325 nm excitation in a dual LRETprobe assay using Terbium chelate as a donor and ROX as an acceptor weredisplayed. It is again very clear that at certain emission wavelengthsthe signal-to-background ratio approaches infinity. As seen from theinsert of FIG. 8 b, for ROX-labeled acceptor beacon, the optimaldetection wavelength is around 614 nm. Although the fluorescenceemission due to energy transfer was very low, these results neverthelesssuggest that there is a significant potential for use of lanthanidedonors with dual energy transfer molecular beacons.

To determine the possible detrimental effect of small gap size betweendonor and acceptor probes on LRET, the spacing between theTerbium-labeled donor probe and the Cy3 or ROX-labeled acceptor beaconwas varied from 3 to 9 bases. It was found that the detectedfluorescence intensity is not sensitive to the gap spacings tested,i.e., with a spacing of 3, 4, 5, 6 and 9 bases, the signal levels weresimilar (data not shown), suggesting that the possible detrimentaleffect was negligible when both probes hybridized to the target with arelatively small gap spacing. Using Equation (1), it is readily shownthat, with the gap spacing varying from 3 to 9 bases, the energytransfer efficiency does not change much, since the Förster distance R₀for the Terbium/Cy3 LRET pair is large (6.12 nm) (Selvin, 2002). Forexample, when R in Equation (1) increases from 1 nm (˜3 bases) to 2 nm(˜6 bases) and to 3 nm (˜9 bases), the energy transfer efficiency E onlydecreases by 0.12% and 1.37%, respectively.

Due to the narrow emission peaks exhibited by lanthanide dyes, and theuse of time-resolved fluorescence detection, it is not necessary toinclude a quencher molecule in the acceptor molecular beacons, althoughthe stem-loop hairpin structure of the beacon may still be beneficial.However, when the detection of point mutations is not involved, the useof linear LRET pairs of oligonucleotide probes is attractive owing toits potential in reducing cost while having comparable performance. Todemonstrate the concept, donor oligonucleotide probes labeled with aEuropium chelate at its 3′-end, and acceptor oligonucleotide probeslabeled at its 5′-end with a Cy5 fluorophore were synthesized, andin-solution hybridization and time-resolved emission detection assayswere performed. The resulting emission spectra are displayed in FIG. 9.Similar to the results obtained using Terbium-chelate donors, at 325 nmexcitation, the emission spectrum of Europium donor bound to targetshowed several peaks within the range of 550 nm to 750 nm, asdemonstrated by curve (1). The fluorescence emission of the Cy5-labledacceptor probe due to probe/target hybridization is again almost zero(curve (2)). When both donor and acceptor probes hybridized to the sametarget, there was a sensitized emission of the acceptor due to LRET, asshown by curve (3). Evidently, at certain wavelengths (such as 670 nm),the background signal due to degraded donor and acceptor probes becomesvery low, leading to a high signal-to-background ratio. It was foundthat with the DTPA-cs124 chelate used in this study, the LRET probepairs with a Terbium donor performs better than the LRET probe pair witha Europium donor and a Cy5 acceptor.

Although conventional molecular beacons can in theory detect mRNAtranscripts in living cells, conditions within the intracellularenvironment can limit their utility in cellular imaging of geneexpression. Specifically, molecular beacons bound to target mRNAs cannotbe distinguished from those degraded by nucleases, or destabilized dueto interactions with proteins. Here we report a dual molecular beaconmethod that combines the advantages of molecular beacons with two-proberesonance energy transfer methods. Conventional and time-resolvedfluorescence spectroscopy studies indicate that dual FRET molecularbeacon pairs are capable of distinguishing between bound and degradedbeacons with improved signal to background than previous methods.Moreover, with a lanthanide chelate as the donor dye, thesignal-to-background ratio can be extremely high at certain wavelengths.

These features are especially important in the detection andquantification of gene expression in living cells where false-positivesignals due to probe degradation and interaction with DNA bindingproteins must be distinguished from the ‘true’ signal that results fromprobe/target binding. We envision widespread applications of the dualenergy transfer molecular beacon methods in laboratory and clinicalstudies of gene expression in living cells, tissues and even animalsusing single- or multi-photon microscopy, time-resolved fluorescencemicroscopy, and fluorescence endoscopy. For example, it is plausible touse this methodology for the specific and sensitive detection of theexpression of oncogenes and tumor-suppresser genes in living cells,potentially making it a very simple and effective clinical tool for theearly detection and diagnosis of malignancy.

When using the dual LRET molecular beacons for gene detection andquantification examples of chelates that can be employed includeDTPA-cs124, BCPDA(4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid)and BHHCT(4,4-bis(1,1,1,2,2,3,3-heptafluoro-4,6-hexanedion-6-yl)-chlorosulfo-o-terphenyl(Evangelista et al., 1988; Lopez et al., 1993; Yuan et al., 1998; Suedaet al., 2000; Cooper and Sammes, 2000).

Another improvement is to use two photon excitation instead ofone-photon excitation. So far, all the dual FRET beacon/targethybridization assays performed were based on a one-photon excitationsource (Xenon flash lamp). However, because of the overlappingexcitation-emission spectrums of the organic donor and acceptormolecules for FRET, it is often difficult to excite the donor withoutalso directly exciting the acceptor. For example, as demonstrated inFIG. 10 a, the maximum excitation of a donor Fam molecule occurs at ˜500nm, but at the same wavelength an acceptor Cy3 molecule is also excitedto 25% of its maximum. Therefore, if an acceptor beacon is degraded bynucleases and the fluorophore is separated from the dabcyl quencher itwill be excited, giving a false-positive signal. Ideally, free acceptorfluorophores should be minimally excited by the excitation source andthe only ones that are excited would be those due to FRET when both thedonor and acceptor beacon are bound to the same target.

One strategy to minimize the direct excitation of the acceptorfluorophore is to use a two-photon excitation source. Two-photonexcitation cross-sections of fluorophores do not necessarily follow thesame trends as one-photon excitation spectrums. For example, as shown inFIG. 10 b, although the maximum excitation of Cy3 occurs at higherwavelengths than Fam when one-photon excitation is used, with two-photonexcitation the Cy3 fluorophore is actually excited at lower wavelengths.Further, when a donor Fam molecule is maximally excited at ˜790 nm, theCy3 acceptor is only excited about 4% of its maximum. This alone is morethan a 6-fold reduction in the direct excitation of the Cy3 acceptorcompared with one-photon excitation. Two-photon excitation also has theadvantage of reduced photo-bleaching, reduced background fluorescencefrom scattering, and the ability to penetrate deeper into biologicaltissue than one-photon excitation. Therefore, two-photon excitation ispotentially a powerful approach in dual FRET molecular beacon studies.

Example 2

Shared Stem Nucleic Acid Probes

Oligonucleotide Synthesis. Oligonucleotide probes and targets weresynthesized using standard phosphoramidite chemistry on an AppliedBiosystems model 394 automated DNA synthesizer (Foster City, Calif.).Molecular beacons were purified using a 2-step reverse phase (RP) plusion-exchange (IE) high performance liquid chromatography (HPLC) on aWaters Model 600E HPLC system (Millipore Corp., Milford, Mass.). ForRP-HPLC purification, oligonucleotides were loaded on a Hamilton PRP-1column and eluted with a linear 5% to 50% acetonitrile gradient in 0.1 Mtriethyl-ammonium acetate (TEAA) pH 7.2 over 40 minutes. Theoligonucleotides were additionally purified by IE-HPLC using a Source™column (Amersham Pharmacia Biotech, Piscataway, N.J.) and eluted with alinear 0% to 50% 1 M LiCl gradient in 0.1 M Tris pH 8.0 over 40 minutes.Unmodified (target) oligonucleotides were purified using polyacrylamidegel electrophoresis. All oligonucleotides were synthesized at IntegratedDNA Technologies, Inc. (Coralville, Iowa).

Molecular Beacon Design. Two types of molecular beacons were designedand synthesized; both contain target-specific probe sequencecomplementary in antisense orientation to exon 6 of the human GAPDHgene, a Cy3 fluorophore at the 5′-end, and a dabcyl quencher at the3′-end. As illustrated in FIG. 11 a, one type follows the conventionaldesign of molecular beacons in that the target-specific probe domain wascentrally positioned between two complementary arms that form the stem;the sequence of these arms were independent of the target sequence.Shared-stem molecular beacons, on the other hand, were designed to haveone arm of the stem complementary to the target sequence, as shownschematically in FIG. 11 b. In both cases, the probe length L_(p) isdefined as the portion of the molecular beacon that is complementary tothe target, and the stem length L_(s) is the number of bases of eachcomplementary arm. All the molecular beacons had L_(p)=19 bases (seeTable 3). Conventional molecular beacons were synthesized with L_(s)=4,5 and 6 bases. The shared-stem molecular beacons were synthesized withL_(s)=4, 5 and 7 bases. As illustrated by FIG. 12 a, a 6-base stem maynot be synthesized because the shared-stem molecular beacon sequence isconstrained, i.e., part of the arm sequence that makes up the 6-basestem is predetermined since the 5′-end of the shared-stem molecularbeacon must complement the target sequence and the 3′-stem is createdsolely to complement the 5′-stem sequence. This inadvertently forces anadditional base pairing in the stem. It should be noted that the stemsequence of a shared-stem molecular beacon is not adjustable since onearm of the stem is designed to complement the target. This limitationoften precludes the design of certain stem/probe length combinations, asdemonstrated in FIG. 12 b for a molecular beacon with a probe length of18 bases and a stem length of 4 bases. Five target oligonucleotides werealso synthesized, one wild-type and four with mismatches at assortedlocations, as shown in Table 3.

TABLE 3 The Design of Probes and Target Oligonucleotides Name Sequence(5′-3′) Notes Shared-stem 19/4 Cy3-GAGTCCTTCCACGATACCActc-Dabcyl Probe19/Stem 4 SEQ ID NO:13 Shared-stem 19/5Cy3-GAGTCCTTCCACGATACCAgactc-Dabcyl Probe 19/Stem 5 SEQ ID NO:14Shared-stem 19/7 Cy3-GAGTCCTTCCACGATACCAggactc-Dabcyl Probe 19/Stem 7SEQ ID NO:15 Conventional 19/4 Cy3-cctcGAGTCCTTCCACGATACCAgagg-DabcylProbe 19/Stem 4 SEQ ID NO:16 Conventional 19/5Cy3-ctgacGAGTCCTTCCACGATACCAgtcag-Dabcyl Probe 19/Stem 5 SEQ ID NO:17Conventional 19/6 Cy3-ctgagcGAGTCCTTCCACGATACCAgctca-Dabcyl Probe19/Stem 6 SEQ ID NO:18 Target WT ACTTTGGTATCGTGGAAGGACTCATGA Perfectmatch SEQ ID NO:19 Target A ACTTTGGTATCGTGGAAGGAaTCATGA Single mismatchSEQ ID NO:20 Target B ACTTTGGTATCGTaGAAGGACTCATGA Single mismatch SEQ IDNO:21 Target C ACTTTGGTATCGTaGAAGGAaTCATGA Double mismatch SEQ ID NO:22Target D ACTTTGGTATCGTaaAAGGACTCATGA Double mismatch SEQ ID NO:23 (1)Molecular Beacons: Lower case = bases added to create stem domains.Upper case = probe-target hybridizing domains. Upper case bold = basesparticipating in both stem hairpin and target binding (2) Targets:Underscore = 19 base sequence complementary to beacons. Lower case bold= mismatch bases in targets

Equilibrium Analysis. Molecular beacons in the presence of target wereassumed to exist in three phases: 1) as duplex with target, 2) asstem-loop hairpin, and 3) in random coil conformation. Dissociationconstants describing the transition between these phases were determinedby analyzing the thermal denaturation profile of molecular beacons inthe presence and absence of target (Bonnet et al., 1999). Denaturationprofiles were obtained by recording the fluorescence intensity of a 50μL solution containing 200 nM of molecular beacon in the presence of 0to 20 μM of target at temperatures ranging from 5° C. to 95° C.Specifically, the temperature of the hybridization solution was broughtto 95° C. and reduced by 1° C. increments to 5° C. The temperature wasthen raised with 1° C. increments back to 95° C. to ensure that thesolution reached equilibrium and no hysteresis had occurred. Thetemperature was held at each temperature increment for ten minutes andfluorescence

was measured for the final 30 seconds. The fluorescence intensity ofeach test solution was adjusted to correct for the intrinsic variance offluorescence over temperature. Each thermal denaturation assay wasperformed in hybridization buffer containing 10 mM Tris, 50 mM KCl, and5 mM MgCl₂.

The fluorescence intensity data describing the thermal denaturationprofile of each molecular beacon and molecular beacon-target duplex wasused to determine the respective dissociation constant as described inBonnet et al. 1999. Specifically, dissociation constants K₁₂characterizing the transition between phase 1 (bound to target) andphase 2 (closed beacon) of molecular beacons were obtained for allbeacon-target pairs and for all molecular beacons in the absence oftarget. Further, the dissociation constants K₁₂ were used to determinethe changes in enthalpy (ΔH₁₂) and entropy (ΔS₁₂) associated with eachbeacon-target duplex. The errors calculated for the thermodynamicparameters signify a 95% confidence interval.

Molecular Beacon Specificity. The fraction of molecular beacons bound totarget, α, was calculated for each molecular beacon-target pair as afunction of temperature. All calculations utilized the thermodynamicparameters, enthalpy change ΔH₁₂ and entropy change ΔS₁₂, obtained fromthe thermal denaturation profiles for each beacon-target duplex

$\begin{matrix}{\frac{\alpha}{\left( {1 - \alpha} \right)\left( {\eta - \alpha} \right){\hat{B}}_{0}} = {\mathbb{e}}^{{({{- \Delta}\;{H_{12}/R}\;\theta})} + {({\Delta\;{S_{12}/R}})}}} & (2)\end{matrix}$where θ is the temperature in Kelvin, R is the gas constant, η=T₀/B₀,{circumflex over (B)}₀=B₀/c₀, T₀ and B₀ are respectively initialconcentration of target and beacons, and c₀ is the unit concentration 1M(Ratilainen et al., 1998). The value of α was calculated for eachmolecular beacon-target pair as a function of temperature for samplescontaining B₀=200 nM of molecular beacon and T₀=400 nM of target. Themelting temperature θ_(m) is defined as the temperature at which half ofthe molecular beacons are bound to target, i.e., α=0.5.

Kinetic Analysis. A SPEX fluorolog-2 spectrofluorometer with an SFA-20rapid kinetics stopped-flow accessory and a temperature/trigger module(SFA-12) was used to measure molecular beacon-target binding kinetics.Specifically, the fluorescence intensity emitted from a rapidly mixedsolution containing 250 nM molecular beacons and 2.5 μM targets wasrecorded over time for each molecular beacon-target pair. Thehybridization reaction was assumed to obey the second order reactionkinetics

$\begin{matrix}{{{B + T}\overset{k_{1}}{\underset{k_{2}}{\underset{\leftarrow}{\rightarrow}}}D},\mspace{14mu}{\frac{\mathbb{d}\lbrack D\rbrack}{\mathbb{d}t} = {{{k_{1}\lbrack B\rbrack}\lbrack T\rbrack} - {k_{2}\lbrack D\rbrack}}}} & (3)\end{matrix}$where [B], [T] and [D] are the concentrations of unbound molecularbeacon, unbound target, and molecular beacon-target duplex,respectively; k₁ is the on-rate and k₂ the off-rate of molecularbeacon-target hybridization. The exact solution of Equation 3 gives

$\begin{matrix}{{1 - \frac{\left\lbrack {D\mspace{11mu}(t)} \right\rbrack}{\left\lbrack D_{eq} \right\rbrack}} = {{\mathbb{e}}^{\Delta\; k_{1}t}\left\lbrack {1 - {\lambda\frac{\left\lbrack {D\mspace{11mu}(t)} \right\rbrack}{\left\lbrack D_{eq} \right\rbrack}}} \right\rbrack}} & (4)\end{matrix}$where Δ=√{square root over ((B₀+T₀+K₁₂)²−4B₀T₀)},[D_(eq)]=(B₀+T₀+K₁₂−Δ)/2, λ=[D_(eq)]²/B₀T₀, and K₁₂=k₂/k₁ is thedissociation constant discussed above. Since the concentration ofmolecular beacon-target duplex is unknown at any given time, it wasassumed that (F(t)−F_(o))/(F_(eq)−F_(o))=[D(t)]/[D_(eq)]where F(t) isthe fluorescence intensity at time t, F_(o) is the initial fluorescenceintensity, and F_(eq) is the fluorescence intensity as t→∞. In order toobtain the on-rate k₁ based on the fluorescence measurement, twodifferent curve-fitting schemes were used. The first utilized aleast-square method by fitting a straight line to a logarithmic form ofEquation 4,

$\begin{matrix}{{\frac{1}{\Delta}\ln\mspace{11mu}\left( {1 - \frac{\left\{ {{F\mspace{11mu}(t)} - F_{o}} \right\}}{\left\{ {F_{eq} - F_{o}} \right\}}} \right)} = {{\frac{1}{\Delta}\ln\mspace{11mu}\left( {1 - {\lambda\frac{\left\{ {{F\mspace{11mu}(t)} - F_{o}} \right\}}{\left\{ {F_{eq} - F_{o}} \right\}}}} \right)} - {k_{1}t}}} & (5)\end{matrix}$with a slope equal to k₁. Alternatively, a non-linear least-squaremethod was used to determine the value of k₁ from Equation 4 directly.The results obtained using these two approaches were compared.

Thermal Analysis. To better understand how the performance ofshared-stem molecular beacons differ from that of the conventionalmolecular beacons, thermodynamic parameters of these two types ofmolecular beacon were obtained and compared. In particular, the enthalpyand entropy changes ΔH₁₂ and ΔS₁₂ describing the phase transitionbetween bound-to-target and stem-loop conformations were determined forconventional and shared-stem molecular beacons using van't Hoff plots.As demonstrated in FIG. 13, these plots display the inverse of meltingtemperature 1/θ_(m) as determined by R ln(T₀−0.5B₀) shown as theordinate. Since at melting temperature,

$\begin{matrix}{{R\;\ln\mspace{11mu}\left( {T_{0} - {0.5B_{0}}} \right)} = {{{- \Delta}\; H_{12}\frac{1}{\theta_{m}}} + {\Delta\; S_{12}}}} & (6)\end{matrix}$the slope of the fitted straight line of each curve in FIG. 13represents the enthalpy change−ΔH₁₂ and the y-intercept represents theentropy change ΔS₁₂. It was found that in general, the shared-stemmolecular beacons have a higher melting temperature, i.e., they formmore stable probe/target duplexes than conventional molecular beacons.The changes of enthalpy and entropy for all the molecular beacon-targetcombinations tested are summarized in Table 4.

TABLE 4 Changes in Enthalpy and Entropy of Conventional and Shared-stemMolecular Beacons in the Presence of Target Conventional MolecularBeacons Shared-stem Molecular Beacons Probe Stem −ΔH ΔS Stem −ΔH ΔSTarget Length Length (kJ/mol) (kJ/mol · K) Length (kJ/mol) (kJ/mol · K)WT 19 4  823 ± 168  2281 ± 489 4  862 ± 116 2383 ± 336 A 19 4 577 ± 62 1595 ± 184 4 708 ± 36 1967 ± 106 B 19 4 527 ± 27 1471 ± 79 4 586 ± 541628 ± 161 C 19 4 472 ± 23 1336 ± 70 4 478 ± 49 1340 ± 148 D 19 4 480 ±38  1352 ± 115 4 521 ± 87 1461 ± 262 WT 19 5 649 ± 28 1784 ± 83 5 690 ±16 1887 ± 46  A 19 5 418 ± 23 1133 ± 70 5 446 ± 20 1205 ± 59  B 19 5 385± 24 1055 ± 73 5 391 ± 24 1057 ± 72  C 19 5 324 ± 17  901 ± 54 5 369 ±57 1025 ± 175 D 19 5 291 ± 25  790 ± 76 5 319 ± 28 861 ± 85 WT 19 6 467± 16 1265 ± 48 7 413 ± 10 1096 ± 28  A 19 6 404 ± 25 1105 ± 76 7 370 ±13 998 ± 38  B 19 6 380 ± 26 1055 ± 79 7 351 ± 39 956 ± 117 C 19 6 367 ±19 1058 ± 61 7 260 ± 30 707 ± 93  D 19 6 373 ± 29 1065 ± 91 7 245 ± 25653 ± 79 

To minimize the number of independent variables involved in controllingprobe/target hybridization, all the molecular beacons were designed tohave identical probe sequences. Further, for molecular beacons with astem length of 5 bases and a probe length of 19 bases, the stem sequenceof the conventional molecular beacons was chosen such that energeticallythe stem was similar to that of the shared-stem molecular beacons. Thefree energy changes were calculated using nearest neighborapproximations (Zucker 2000).

The difference in thermodynamic behavior between conventional andshared-stem molecular beacons can be understood in terms of the abilityof the flanking arms to interact with each other. With shared-stemmolecular beacons, once part of the stem (one arm) is bound to thetarget, it is less likely to interact with its complementary arm,resulting in a more stable probe/target duplex. In contrast, the arms ofa conventional molecular beacon do not bind to the target and are thusmore likely to interact with each other as driven by thermal energy,increasing the tendency of forming a closed molecular beacon bydissociating from the target. Not surprisingly, the stem length of amolecular beacon influenced the equilibrium state of both theshared-stem and conventional beacons in the presence of target. As shownin FIG. 14, as the stem length was increased from 4 to 6 bases,conventional molecular beacons were found to dissociate from targetmolecules more readily. A very similar trend was true for shared-stemmolecular beacons (data not shown). This indicates that hybridization isless favorable for molecular beacons with longer stems.

The changes in enthalpy and entropy that control the dissociation ofconventional and shared-stem molecular beacons from targets withmismatches were also determined (see Table 4). It was found thatshared-stem molecular beacons formed more stable duplexes with each ofthe target molecules tested. However, as displayed in FIG. 15, whenpoint mutations were present in the target oligonucleotide, both typesof molecular beacons dissociated from their targets more readily. Themagnitude of change depended on both the number of mismatches and theirlocation. Compared with the wild-type target, a point mutation near thecenter of the probe-binding domain (Target B) was found to have a largereffect on molecular beacon dissociation than a mutation near the end ofthe probe-binding domain (Target A). As expected, two point mutations(Targets C and D) on a target had a more profound effect on thedissociation of molecular beacons from targets than that with one pointmutation.

Melting Temperature. To further elucidate the effect of molecular beaconstructure on the stability of the probe-target duplex, the meltingtemperatures θ_(m) for conventional and shared-stem molecular beaconswith a probe length of 19 bases and stem lengths ranging from 4 to 7bases were compared, as shown in FIG. 6. It was found that conventionalmolecular beacons had lower melting temperatures than shared-stemmolecular beacons for each of the stem lengths considered; however, bothtypes of molecular beacons exhibited similar trends. Specifically, themelting temperature progressively decreased as the stem lengthincreased. In fact, it appears that the melting temperature would bequite low for conventional molecular beacons with a probe length of 19bases and a stem length of 7 bases or greater. This is because that withlong free arms of the stem a bound molecular beacon is very easy todissociate from the target and form a stable hairpin structure even atlow temperatures.

Molecular Beacon Specificity. Melting curves that display the fractionof molecular beacons in duplex form, α, as a function of temperaturewere obtained for each molecular beacon and probe-target pair. Asdemonstrated in FIG. 17 a, the difference in melting temperature θ_(m)(i.e., temperature at α=0.5) between beacon/wild-type-target andbeacon/mutant-target duplexes was found to be slightly larger forconventional molecular beacons compared with corresponding shared-stemmolecular beacons. Further, the difference in the fraction of molecularbeacons bound to wild-type target and mutant target,α_(WT)−α_(Target B), as a function of temperature was found to besimilar for conventional and shared-stem molecular beacons, although themaximum value of α_(WT)−α_(Target B) is slightly higher for the former,as shown in FIG. 15 b. The conventional molecular beacons was also foundto maintain a value of α_(WT)−α_(Target B)>0 over a slightly broaderrange of temperatures than shared-stem molecular beacons, but again thedifferent is very small. This implies that the conventional molecularbeacons may exhibit only a slightly higher specificity than shared-stemmolecular beacons.

The effect of stem length on molecular beacon specificity was also foundto be similar for conventional and shared-stem molecular beacons.Specifically, the curves in FIG. 18 demonstrate that, as the stem lengthis increased the heightened competition between a unimolecular reactionand bimolecular hybridization broadens the transition between bound andunbound states. This results in an improved ability to discriminatebetween targets over a wider range of temperature but lowers the maximumdifference in the fraction of beacons bound to wild-type and mutanttargets.

Kinetic Analysis. The on-rate of shared-stem and conventional molecularbeacons hybridized to wild-type target as a function of stem length isdisplayed in FIG. 19 a. It is seen that for shared-stem molecularbeacons, an increase in stem length from 4 to 5 bases induced a 5-foldreduction in its on-rate, which was further reduced by 3-fold when thestem length was increased from 5 to 7 bases. In contrast, the on-rate ofconventional molecular beacons only decreased slightly when the stemlength was increased from 4 to 6 bases. It is interesting to note that,with stem lengths of 5 bases or larger, the shared-stem and conventionalmolecular beacons have on-rates differing only by less than a factor of2. However, the shared-stem molecular beacons with a 4-base stemhybridized to wild-type targets four times faster than the correspondingconventional molecular beacons. This large difference in the rate ofhybridization most likely resulted from the variations in the stabilityof the hairpin structure. To further illustrate, the dissociationconstants K₂₃ of the conventional and shared-stem molecular beacons inthe absence of target are shown in FIG. 19 b. Interestingly, there seemsto be a clear correlation between the on-rate of beacon hybridizationand the stability of the stem-loop structure. This is understandablesince K₂₃ represents the transition from hairpin (phase 2) to randomcoiled (phase 3) conformations of molecular beacons, and a higher K₂₃implies that the molecular beacons are easier to open.

Molecular beacons have become a very useful tool for many homogeneoussingle-stranded nucleic acid detection assays due to their ability todifferentiate between bound and unbound states and their improvedspecificity over linear probes. However, to optimize the performance ofmolecular beacons for different applications, it is necessary tounderstand their structure-function relationships. Here is described anew design of molecular beacons, i.e., the shared-stem molecularbeacons, of which the stem-arm nearest the reporter dye participates inboth hairpin formation and target hybridization. In contrast toconventional molecular beacons whose stems are independent of the targetsequence and thus can freely rotate around the probe-target duplex, thisnew design helps immobilize the fluorophores of molecular beacons whenthey hybridize to the target, which is desirable when two molecularbeacons are used in a fluorescence resonance energy transfer (FRET)assay (Tsourkas and Bao 2001). Specifically, with shared-stem molecularbeacons, there is a better control of the distance between the donor dyeon one beacon and the acceptor dye on the other beacon, since therotational motion of the fluorophore-attached stem-arm is constrained,as illustrated in FIG. 11 b. To facilitate the design and applicationof, and to reveal the differences between, shared-stem and conventionalmolecular beacons, we performed a systematic study of the thermodynamicand kinetic parameters that control the hybridization of these molecularbeacons with complementary and mismatched targets.

In general, it was found that compared with shared-stem molecularbeacons, conventional molecular beacons form less stable duplexes withsingle-stranded nucleic acid targets but have a slightly improvedability to discriminate between wild-type and mutant targets. Thedifference in the duplex stability can be explained by thethermal-driven interactions between the two stem-forming arms after themolecular beacon hybridized to a target molecule. Unlike linearoligonucleotide probes, a molecular beacon can have two stableconformations: bound to target, and as a stem-loop hairpin. These twostable states compete with each other, giving rise to an improvedspecificity. The additional freedom inherent in both arms ofconventional molecular beacon increases the likelihood that, driven bythermal fluctuations, these arms will encounter with each other,allowing the molecular beacon to dissociate from the target with ahigher probability. This reduced stability also corresponds to a smallervalue in the free energy difference between bound and unbound states ofthe probe-target duplex. The change in free energy due to any mismatchbetween the probe and target, therefore, will have a more profoundeffect on the preference of the stem-loop hairpin conformation of theconventional molecular beacons, leading to an improved ability todifferentiate between wild-type and mutated targets. However, thisimprovement was found to be marginal.

With any given probe length and sequence, the hybridization kinetics ofmolecular beacons appears to be primarily dependent on the length andsequence of the stem, regardless of whether they are designed in theconventional or shared-stem configuration. Both types of molecularbeacons exhibited comparable hybridization rates when the dissociationconstants describing the thermal fluctuation induced opening of thestem-loop structure, K₂₃, were similar. When the difference in K₂₃ forthe shared-stem and conventional molecular beacons was increased, so wasthe difference in the hybridization on-rate.

In addition to the above-mentioned differences in the behavior ofshared-stem and conventional molecular beacons, the choice of the stemlength is independent of the probe length for conventional molecularbeacons, whereas there are certain constraints on the stem-length andprobe-length combinations in designing the share-stem molecular beacons.This, together with the dependence of the thermodynamic and kineticproperties on the probe and stem lengths demonstrated in this study,should be considered in the design of molecular beacons for specificapplications.

Example 3

K-ras and Survivin Detection

It is well established that cancer cells develop due to geneticalterations in oncogenes and tumor suppressor genes and abnormalities ingene expression that provide growth advantage and metastatic potentialto the cells. A critical step in diagnosing and treating cancer in itsearly stages is to detect cancer cells based on the genetic alterations.An important example is pancreatic cancer, the fifth most fatal cancerin the US. Only 12% of patients diagnosed with pancreatic cancer cansurvive for one year; the 5-year overall survival rate is approximately3-5%. The main reason for the poor prognosis of pancreatic cancer isthat very few of these cancers can be found early. Current clinicaldiagnostic procedures such as CT-scan and endoscopic retrogradecholangiopancreatography (ERCP) have a low sensitivity in detectingpancreatic tumors less than 2 cm in size. In spite of the extensivebiomedical research efforts during the last few decades, over 90% of thepatients with pancreatic cancer have already undergone local and/ordistant metastases by the time of diagnosis, often making it too late tocure. Therefore, it is extremely important to detect pancreatic cancerin its early stages based on molecular markers rather than the size ofthe tumor.

A novel way of achieving early detection of cancer is to identify cancercells through detection of mRNA transcripts that exist in cancer cellsbut not in normal cells. Here is demonstrated the dual-FRET molecularbeacons of the present invention for the early detection of cancercells.

K-ras is one of the most frequently mutated genes in human cancers. Amember of the G-protein family, K-ras is involved in transducinggrowth-promoting signals from the cell surface. Point mutations of K-rasare found in 80-100% of pancreatic, 40-60% of colon, and 25-50% of lungadenocarcinomas, suggesting that mutant K-ras is a sensitive marker forpancreatic cancer detection. Further, K-ras mutations occur almostexclusively in three hot spots (codons 12, 13 and 61). Most of them areconcentrated at codon 12, which facilitates the design of molecularbeacons. Since K-ras mutations occur very early in the development ofpancreatic cancer, assays targeting K-ras mutations can lead to earlydetection of pancreatic carcinomas. Other oncogenes and tumor-suppressorgenes involved in pancreatic cancer include p53, p16, MADH4, DPC4,BRCA2, MKK4, STK11, TGFBR1 and TGFBR2.

There is increasing evidence recently suggesting that survivin, one ofthe inhibitor of apoptosis proteins (IAPs), is a good tumor marker forseveral types of cancers. Survivin is normally expressed during fetaldevelopment but not in most normal adult tissues. However, high levelsof survivin are detected in many human cancer types and transformedhuman cells. In particular, a recent study has demonstrated the presenceof survivin in 77% (20 out of 26 cases) of pancreatic duct celladenocarcinomas by immunohistochemistry, immunoblotting and RT-PCRassays. The results from this study also suggested that the expressionof survivin is present in early stages of neoplastic transition inpancreatic cancer cells. However, expression of survivin was notdetected in pancreatic tissues obtained from 5 normal persons and 12patients with chronic pancreatitis, nor was it found in inflammatorycells around tumor cells. The absence of survivin expression in normalpancreas, pancreatic tissue of chronic pancreatitis patients and othernormal tissues makes it an ideal molecular marker for the detection ofpancreatic cancer cells. Although molecular beacons can be designed totarget alterations of many oncogenes and tumor-suppressor genes, in theproposed study we opt to focus on the detection of K-ras mutations andthe expression of survivin in pancreatic cells.

It has been shown that K-ras mutations can be detected in blood,pancreatic juice and pancreatic tissue samples of pancreatic cancerpatients using DNA purification and mutant-enriched PCR followed bysingle strand conformation polymorphism (SSCP), restrictionfragment-length polymorphisms (RELP), or allele-specificoligodeoxynucleotide hybridization (ASOH). Although identification ofK-ras mutations by PCR is a fairly sensitive molecular approach, theprocedures for PCR and subsequent assays are very time-consuming, makingthem difficult to become clinical procedures. Furthermore, detection ofK-ras mutations in DNA from peripheral blood or pancreatic juice aloneis not sufficient for diagnosis of pancreatic cancer since it lacks thespecificity for the determination of the cellular origin of the K-rasmutation. A better way to detect pancreatic cancer is to use nucleicacid probes of the present invention to detect K-ras mutations in cancercells directly. Utilization of prior nucleic acid probes to detect K-rasmutations in PCR products of DNA samples isolated from lung cancers hasbeen reported and the specificity has been established. However, to datethe use of nucleic acid probes for the detection of mutant K-ras mRNA inintact tumor cells has not been reported. One advantage of using themolecular beacons approach is that a cocktail of multiple such probescan be delivered into cells for different molecular markers of cancer.

An important issue in detecting K-ras mutations in cells is that as asignaling protein the expression level of K-ras mRNA may not be veryhigh (<1,000 copies per cell), even in cancer cells. Further, thesecondary structures of K-ras and survivin mRNAs may influence thebinding between nucleic acid probes of the present invention and thetargets. Thus, it is preferred to optimize the design of molecularbeacons and the beacon delivery conditions so that high detectionspecificity and sensitivity can be achieved. By routinely combining thenucleic acid probes of the present invention approach withhigh-sensitivity fluorescence microscopy, it will likely be possible todetect as few as 10 copies of mRNA per cell.

To further examine probe-target hybridization and energy transferbetween nucleic acid probes of the present invention, dual-FRETmolecular beacons were designed and synthesized. Specifically, themolecular beacons were designed to target human GAPDH mRNA. The donorproes were synthesized with a 6-FAM donor fluorophore (D) at the 3′ endand a Dabcyl quencher (Q) at the 5′ end. Similarly, acceptor probes weresynthesized with a Cy3 acceptor fluorophore (A) at the 5′ end and aDabcyl quencher (Q) at the 3′ end (see Table 5 below). The donor andacceptor beacons are chosen such that they are complementary to part ofthe target sequence. The loop portion, therefore, is 13 bases in length.The synthetic targets mimicking the GAPDH IVT RNA exon 6/exon 7 junctionare designed so that the gap between the two beacons hybridizing on thesame target is respectively 3, 4, 5 or 6 bases, with 4-base gap beingthat of the wild-type. All the nucleic acid probes of the presentinvention were synthesized at Integrated DNA Technologies (IDT, Inc).

As illustrated above, thermodynamics and binding kinetics of molecularinteractions underlies the design of the nucleic acid probes of thepresent invention. The free energy difference between keeping thestem-loop structure and forming the probe-target duplex is the maindriving force for hybridization. The nucleic acid probes of the presentinvention can have three different phases in solution: hairpin, randomcoil, and probe-target duplex. The relative portions of these phasesdepend on the structure of the probe, probe and target concentrations,solution chemistry, sequences of the probe and target, and temperature.For example, if the stem length is too large, it will be difficult forthe stem-loop probe to open upon hybridization. On the other hand, ifthe stem length is too small, a large fraction of probes may open due toBrownian forces. Similarly, relative to the stem length, a longer probemay lead to a lower dissociation constant, however, it may also reducethe specificity, since the relative free energy change due to one-basemismatch would be smaller.

To further establish the structure-function relationships of probes, onecan for example routinely design and synthesize a series of dual-FRETprobes for targeting different K-ras codon 12 mutations, such as shownin Table 5. For each pair of donor and acceptor beacons, the donorbeacon will contain one of the most common K-ras point mutations inpancreatic cancer such as GGT-GAT transitions (Gly to Asp) or GGT-GTT(Gly to Val), GGT-CGT (Gly to Arg), GGT-TGT (Gly to Cys) transversions.The same acceptor beacon can used with all donor beacons havingdifferent mutated sequences. As an example, a specific beacon design fornucleic acid probes of the present invention has a probe length of 21nucleotides, a stem length of 4 nucleotides, and a gap size of 4nucleotides between the donor and acceptor beacons bound on the sametarget. One can routinely examine the effect of beacon structure onhybridization rate and specificity by varying: 1) probe length of 17, 19and 21 bases; 2) stem length of 4 and 5 bases; 3) gap sizes of 4 and 5bases between donor and acceptor beacons along the target mRNA. Fordifferent parameter combinations, kinetic and thermodynamic modelingdescribed above will be performed to build the analysis of theexperimental data.

TABLE 5 Design of Molecular Beacons for K-ras Codon 12 MutationWild-type K-ras (Bases 1-78)  1 ATGACTGAAT ATAAACTTGTGGTAGTTGGA GCTGGTGGCG 41 TAGG caag AG TGCCTTGACG ATACAGCTAA TTCAGAAT  SEQ ID NO:27 Dual-FRET Molecular Beacons Donor Beacon:5′-Dabcyl-AGTGCGCTGTATCGTCAAGGCACT-6-Fam-3′ SEQ ID NO:28 AcceptorBeacon: 5′-Cy3-CCTACGCCATCAGCTCCGTAGG-Dabcyl-3′ Mut: GGT-GAT SEQ IDNO:29 Acceptor Beacon: 5′-Cy3-CCTACGCCAACAGCTCCGTAGG-Dabcyl-3′ Mut:GGT-GTT SEQ ID NO:30 Acceptor Beacon:5′-Cy3-CCTACGCCACGAGCTCCGTAGG-Dabcyl-3′ Mut: GGT-CGT SEQ ID NO:31Acceptor Beacon: 5′-Cy3-CCTACGCCACAAGCTCCGTAGG-Dabcyl-3′ Mut: GGT-TGTSEQ ID NO:32

To further increase the detection sensitivity, for example, one cansynthesize nucleic acid probes of the present invention to target asecond cancer marker, such as survivin, which is expressed in pancreaticcarcinomas but not in normal tissues.

To demonstrate the specificity of molecular beacons targeting K-raspoint mutations, in-solution hybridization studies are carried out bymixing the donor nucleic acid probes of the present invention withrespectively wild-type K-ras mRNA targets, the corresponding mutatedK-ras mRNA targets and survivin targets at different probe/targetconcentrations. Thermal denaturation profiles are generated and thecorresponding transition temperatures obtained. Since the detectionspecificity depends on the initial concentrations of probes and targets,the results of this study not only demonstrate the detection specificitybut also provide guidelines for optimizing beacon delivery conditions.Furthermore, stopped-flow studies of the hybridization kinetic rates areperformed with each nucleic acid probe of the present invention designand each target type. This helps select the optimal structure of thenucleic acid probes of the present invention with the best possiblecombination of specificity and rate of hybridization.

To address potential issues with secondary structures of the mRNAs,synthetic targets with lengths three to four times the probe length areused in in-solution studies. To further utilize the nucleic acid probesof the present invention, total mRNAs of survivin and the mutant K-rasare isolated from pancreatic cancer cells known to carry them,amplified, and in-solution hybridization assays are performed todetermine the extent of binding between the nucleic acid probes of thepresent invention and these mRNAs.

As mentioned earlier, in a cellular environment, nucleic acid probes canbe degraded by cytoplasmic nucleases. To address this issue, the presentinvention provides probes synthesized with structural modifications suchas 2′-O-methyl ODNs. Methylated-probe/target duplexes are more stablethan DNA-probe/target duplexes, and methylated nucleic acid probes ofthe present invention hybridize to RNA targets faster than DNA probes.After delivery into primary human dermal fibroblast (HDF) cells, thefluorescence signal of unmodified molecular beacons with a random DNAsequence and methylated nucleic acid probes of the present inventionwith the same sequence are monitored over time to determine how longthese probes can survive in a cellular environment.

To determine the specificity of the molecular beacons approach fordetecting K-ras mutations in pancreatic cancer cells, nucleic acidprobes of the present invention are synthesized to target four differentK-ras codon 12 mutations (GGT-GAT, GGT-GTT, GGT-CGT and GGT-TGT).Delivery, hybridization and imaging assays are carried out usingpancreatic cancer cell lines such as Panc-1 (GGT to GAT), Capan-1 (GGTto GTT), PSN-1 (GGT to CGT), and Miapaca-2 (GGT to TGT) with thecorresponding mutant K-ras mRNAs. Also used is the pancreatic cell lineBXPC-3 as a control which has the ‘wild-type’ K-ras mRNA.

Preliminary studies have shown that the K-ras codon 12 mutation GGT-GATin Panc-1 cell lines has been confirmed. Further confirmation of theother three mutations (GGT-GTT, GGT-CGT and GGT-TGT) in thecorresponding pancreatic cancer cell lines using PCR amplification ofK-ras exon 1 sequence followed by DNA sequencing is routinely performed.The K-ras mRNA concentration in each cell line is also be quantifiedusing real time RT-PCR. During a pilot study, the delivery,hybridization, and buffer conditions for the nucleic acid probes of thepresent invention have been optimized to target mutant K-ras mRNA inPanc-1 cells (with GGT to GAT mutation). As mentioned earlier, theinvention provides a preferred condition for this type of probe of 150nM of the probes in Opti-MEM 1 medium (GIBCO) and incubated at 37° C.for 30 to 60 minutes. It is anticipated that the optimal delivery andhybridization conditions for nucleic acid probes of the presentinvention targeting different mutant K-ras mRNAs may vary from thoseidentified. Nucleic acid probes of the present invention designed foreach specific K-ras mutation are incubated with at least four cell lineswith the optimal delivery condition; these cell lines include the cellline containing the specific K-ras mutation, the ‘wild-type’ K-ras cellline BXPC-3, and two other cell lines with different K-ras mutations.The FRET-induced fluorescence signal in cells can be imaged using aconfocal microscope. The specificity of the present probe detectionmethodology is confirmed when strong fluorescence signal are observedonly in the cell line expressing the corresponding K-ras mutation butnot in BXPC-3 nor other cell lines.

Similar assays are routinely performed to examine the specificity ofsurvivin-targeting nucleic acid probes of the present invention usingpancreatic cancer cell lines expressing different levels of survivingene as well as the normal human fibroblast cells as a control. Forexample, using RT-PCR and Northern blotting, the level of survivinexpression in pancreatic cell line Miapaca-2 ihas been found to be high,in BXPC-3 it is much lower, while in HDF the expression level is almostzero. The probe sequence of the survivin probes is shown in Table 6 andthe steps of the assay are similar to that described above. The tumorcell lines expressing different levels of survivin mRNA and the normalcell line HDF are incubated with the survivin nucleic acid probes of thepresent invention, and the resulting fluorescence are imaged using aconfocal microscope.

TABLE 6 Design of Molecular Beacons for Survivin mRNA Survivin (Bases1-121)  1 A TGGGTGCCCC GACGTTGCCC CCTGCCTGGC AGCCCTTTCT 42 CAAGG accaC CGCATCTCTA CATTCAAGAA CTGGCCCTTC   82 TTGGAGGGCT GCGCCTGCAC CCCGGAGCGGATGGCCGAGG   SEQ ID NO:33 Dual-FRET Molecular Beacons Donor Beacon:5′-Alexa546-CCTTGAGAAAGGGCTGCCCAAGG-Dabcyl-3′ SEQ ID NO:34 AcceptorBeacon: 5′-Dabcyl-CCGCATTGAATGTAGAGATGCGG-Texas Red-3′ SEQ ID NO:35

A critical issue concerning the specificity of detecting pancreaticcancer cells is that both K-ras codon 12 mutations and survivin arebeing expressed in colorectal and lung cancers as well. To assure highspecificity for detecting cancer cells originated from the pancreaticducts, in addition to using nucleic acid probes of the present inventiontargeting survivin and K-ras mutations, a third probe pair for thechymotrypsinogen gene can be synthesized, which is pancreas-specific. Anexample of the design of donor and acceptor probes are respectively:donor probe: 5′-AMCA-ACCTGGATGTTGTCCTCGTCAGGT-dabcyl-3′ SEQ ID NO:36 andacceptor probe:5′-dabcyl-AAGATTGAAGACCTTGGCGATCTT-Diakylaminocoumarin-3′ (underlinedbases are complementary bases to form a stem) SEQ ID NO:37. This nucleicacid probe pair of the present invention will be delivered intopancreatic, lung and colon cancer cell lines as well as the normal humanfibroblast cell line HDF and the resulting fluorescence images recorded.This will assure that only cells originated from pancreatic duct aredetected. All the detection assays with cells are performed at 37° C.The assays determining detection specificity with lanthanide-dye basednucleic acid probes of the present invention are carried out using aSafire monochromator reader (Tecan).

To determine the sensitivity of the probe-based methodology in detectingpancreatic cancer cells, pancreatic cancer and normal cells are mixedwith 1:1,000 (i.e., one cancer cell in 1,000 normal cells), 1:10,000,1:100,000 and 1:1,000,000 ratios and incubate the mixture with thedual-FRET molecular beacons designed for the specific cancer cell lineunder optimized conditions (probe concentration and duration). Afterplacing cells on glass coverslips, FRET-induced fluorescence images ofthe hybridized nucleic acid probes of the present invention in cancercells are obtained using a confocal microscope. Further, a FACS VantageSE cell sorter (Becton-Dickinson) is used to sort out the cancer cellsin the mixture in suspension. The cell sorter, which has a sortingsensitivity of 1:100,000, has three excitation wavelengths: 488 nm, 547nm, and UV. The fluorescence emission due to dual-FRET probes in cellscan be detected by using the proper filter. The dual-FRET probe pairsfor detecting survivin, K-ras mutations and chymotrypsinogen are sodesigned such that the donor dye molecules are respectively excited withlaser at 353 nm, 488 nm and 547 nm. A fluorescence intensity thresholdin the detection is chosen such that the effect of background due toauto-fluorescence of cells and digested nucleic acid probes of thepresent invention is minimized. In this study, the pancreatic cell linesPanc-1, Capan-1, PSN-1 and Miapaca-2 are used as cancer cells, and ahuman dermal fibroblast cell line (HDF) serves as normal cells.

In obtaining images of hybridized molecular beacons in cancer cells,having ultra-sensitive fluorescence measurements is important, sincetypically only a very small number of cancer cells are present in asample. The FACS Vantage Flow Cytometer (cell sorter) will be used dueto its very high detection sensitivity, capability of 5 color analysisand sorting, wide flexibility of excitation wavelengths, and cross beamlaser compensation for separation of overlapping excitation. An imaginganalysis will also be carried out to enhance the results of FRETfluorescence measurements. In this example, in carrying out imagingassays of detection sensitivity, only nucleic acid probes of the presentinvention with organic dye pairs for FRET will be used, since currentlythe Zeiss confocal microscope and the FACS cell sorter do not have animaging capability with time resolved FRET.

This example is based on the rational that detection of tumor markersincluding survivin and mutant K-ras mRNAs in pancreatic duct cells usingdual-FRET nucleic acid probes of the present invention can lead to earlydiagnosis of pancreatic cancer especially in high-risk patients. Itdemonstrates that the novel dual-FRET molecular beacons methodology ofthe present invention has the potential to become a simple clinicalprocedure for early detection of pancreatic cancer with highsensitivity, specificity, signal-to-noise ratio, and efficiency. This isfurther demonstrated with subsequent translational research usingclinical samples. The same methodology can be rountinely extended to theearly detection and diagnosis of other cancers, and the study of geneexpression in live cells relevant to solving other biomedical problems.

REFERENCES

-   Bernacchi, S., and Y. Mely. 2001. Exciton interaction in molecular    beacons: a sensitive sensor for short range modifications of the    nucleic acid structure. Nucleic Acids Res. 29:E62-2.-   Bonnet, G., S. Tyagi, A. Libchaber, and F. R. Kramer. 1999.    Thermodynamic basis of the enhanced specificity of structured DNA    probes. Proc. Natl. Acad. Sci. USA 96: 6171-6176.-   Bonnet, G., Krichevsky, O. and Libchaber, A. 1998. Kinetics of    conformational fluctuations in DNA hairpin-loops. Proc. Natl. Acad.    Sci. USA, 95, 8602-8606.-   Cardullo, R. A., Agrawal, S., Flores, C., Zamecnik, P. C.,    Wolf, D. E. 1998. Detection of nucleic acid hybridization by    nonradiative fluorescence resonance energy transfer. Proc. Natl.    Acad. Sci. USA. 85, 8790-8794.-   Chen, W., Martinez, G. and Mulchandani, A. 2000 Molecular beacons: a    real-time polymerase chain reaction assay for detecting Salmonella.    Anal. Biochem., 280, 166-172.-   Cooper, M., and P. Sammes. 2000. Synthesis and spectral properties    of a new luminescent europium(III) terpyridyl chelate. J. Chem. Soc.    Perkin. Trans. 2:1695-1700.-   de Baar, M. P., Timmermans, E. C., Bakker, M., de Rooij, E., van    Gemen, B. and Goudsmit, J. 2001 One-tube real-time isothermal    amplification assay to identify and distinguish human    immunodeficiency virus type 1 subtypes A, B, and C and circulating    recombinant forms AE and AG. J. Clin. Microbiol., 39, 1895-1902.-   Dirks, R. W., C. Molenaar and H. J. Tanke. 2001. Methods for    visualizing RNA processing and transport pathways in living cells.    Histochem. Cell Biol. 115:3-11.-   Elangovan M, R. N. Day, and A. Periasamy. 2002. Nanosecond    fluorescence resonance energy transfer-fluorescence lifetime imaging    microscopy to localize the protein interactions in a single living    cell. J. Microsc. 205:3-14.-   Evangelista, R. A., A. Pollak, B. Allore, E. F. Templeton, R. C.    Morton, and E. P. Diamandis. 1988. A new europium chelate for    protein labelling and time-resolved fluorometric applications. Clin.    Biochem. 21:173-178.-   Fang, X., J. J. Li, and W. Tan. 2000. Using molecular beacons to    probe molecular interactions between lactate dehydrogenase and    single-stranded DNA. Anal. Chem. 72:3280-3285.-   Femino, A. M, F. S., Fay, K. Fogarty, and R. H. Singer. 1998.    Visualization of single RNA transcripts in situ. Science 280:585-90.-   Goddard, N. L., Bonnet, G., Krichevsky, O., Libchaber, A. 2000.    Sequence dependent rigidity of single stranded DNA. Phys. Rev.    Lett., 85, 2400-2403.-   Hung, S. C., R. A. Mathies, and A. N. Glazer. 1997. Optimization of    spectroscopic and electrophoretic properties of energy transfer    primers. Analytical Biochemistry 252:78-88.-   Ju, J., I. Kheterpal, J. R. Scherer, C. Ruan, C. W. Fuller, A. N.    Glazer, and R. A. Mathies. 1995. Design and synthesis of fluorescent    energy transfer dye-labeled primers and their application for DNA    sequencing and analysis. Analytical Biochemistry 231:131-40.-   Kang J. S., J. R. Lakowicz, and G. Piszczek. 2002. DNA dynamics: a    fluorescence resonance energy transfer study using a long-lifetime    metal-ligand complex. Arch. Pharm. Res. 25:143-150.-   Klostermeier D, and D. P. Millar. 2002. Time-resolved fluorescence    resonance energy transfer: A versatile tool for the analysis of    nucleic acids. Biopolymers 61:159-179.-   Kuhn, H., Demidov, V. V., Coull, J. M., Fiandaca, M. J.,    Gildea, B. D. and Frank-Kamenetskii, M. D. 2002. Hybridization of    DNA and PNA molecular beacons to single-stranded and double-stranded    DNA targets. J Am Chem Soc., 124, 1097-1103.-   Li, J. J., R. Geyer, and W. Tan. 2000. Using molecular beacons as a    sensitive fluorescence assay for enzymatic cleavage of    single-stranded DNA. Nucleic Acids Res. 28:E52.-   Li, M. and P. R. Selvin. 1997. Amine-reactive forms of a luminescent    diethylenetriaminepentaacetic acid chelate of terbium and europium:    attachment to DNA and energy transfer measurements. Bioconjug. Chem.    8:127-132.-   Liu, J., P. Feldman, and T. D. Chung. 2002. Real-time monitoring in    vitro transcription using molecular beacons. Anal. Biochem.    300:40-45.-   Lemmetyinen, H., E. Vuorimaa, A. Jutila, V. M. Mukkala, H. Takalo,    and J. Kankare. 2000. A time-resolved study of the mechanism of the    energy transfer from a ligand to the lanthanide(III) ion in    solutions and solid films. Luminescence 15:341-350.-   Lopez, E., C. Chypre, B. Alpha, and G. Mathis. 1993. Europium(III)    trisbipyridine cryptate label for time-resolved fluorescence    detection of polymerase chain reaction products fixed on a solid    support. Clin. Chem. 39:196-201.-   Marras, S. A., Kramer, F. R. and Tyagi, S. (1999) Multiplex    detection of single-nucleotide variations using molecular beacons.    Genet. Anal., 14, 151-156.8.-   Matsuo, T. 1998. In situ visualization of messenger RNA for basic    fibroblast growth factor in living cells. Biochim. Biophys. Acta    1379:178-184.-   Mergny, J. L., A. S. Boutorine, T. Garestier, F. Belloc, M.    Rougee, N. V. Bulychev, A. A. Koshkin, J. Bourson, A. V. Lebedev,    and B. Valeur. 1994. Fluorescence energy transfer as a probe for    nucleic acid structures and sequences. Nucleic Acids Res. 22:    920-928.-   Mitchell, P. 2001. Turning the spotlight on cellular imaging. Nat.    Biotechnol. 19:1013-1017.-   Molenaar, C., S. A. Marras, J. C. Slats, J. C. Truffert, M.    Lemaitre, A. K. Raap, R. W. Dirks, and H. J. Tanke. 2001. Linear 2′    O-Methyl RNA probes for the visualization of RNA in living cells.    Nucleic Acids Res. 29:E89-9.-   Morrison, L. E., and L. M. Stols. 1993. Sensitive fluorescence-based    thermodynamic and kinetic measurements of DNA hybridization in    solution. Biochemistry. 32:3095-3104.-   Ratilainen, T., Holmen, A., Tuite, E., Haaima, G., Christensen, L.,    Nielsen, P. E. and Norden, B. 1998. Hybridization of peptide nucleic    acid. Biochemistry, 37, 12331-12342.-   Sei-Iida, Y., H. Koshimoto, S. Kondo, and A. Tsuji. 2000. Real-time    monitoring of in vitro transcriptional RNA synthesis using    fluorescence resonance energy transfer. Nucleic Acids Res. 28:E59.-   Sixou, S., F. C. Szoka, Jr., G. A. Green, B. Giusti, G. Zon,    and D. J. Chin. 1994. Intracellular oligonucleotide hybridization    detected by fluorescence resonance energy transfer (FRET). Nucleic    Acids Res. 22:662-668.-   Sokol, D. L., X. Zhang, P. Lu, and A. M. Gewirtz. 1998. Real time    detection of DNA.RNA hybridization in living cells. Proc. Natl.    Acad. Sci. USA 95:11538-11543.-   Sueda, S., J. Yuan, and K. Matsumoto. 2000. Homogeneous DNA    hybridization assay by using europium luminescence energy transfer.    Bioconjug. Chem. 11:827-831.-   Tsourkas, A., M. Behlke, S. Rose, and G. Bao. 2002a. Hybridization    kinetics and thermodynamics of molecular beacons, submitted to    Biophysical J.-   Tsourkas, A., M. Behlke, and G. Bao. 2002b. Structure-function    relationships of shared-stem and conventional molecular beacons,    submitted to Nucleic Acids Res.-   Tsourkas, A. and Bao, G. 2001. Detecting mRNA transcripts using    FRET-enhanced molecular beacons. In Proceedings of the 2001    Bioengineering Conference, ASME BED-Vol. 50, pp. 169-170.-   Tsuji, A., Y. Sato, M. Hirano, T. Suga, H. Koshimoto, T. Taguchi,    and S. Ohsuka 2001. Development of a time-resolved fluorometric    method for observing hybridization in living cells using    fluorescence resonance energy transfer. Biophys J. 81:501-515.-   Tsuji, A., H. Koshimoto, Y. Sato, M. Hirano, Y. Sei-Iida, S. Kondo,    and K. Ishibashi. 2000. Direct observation of specific messenger RNA    in a single living cell under a fluorescence microscope. Biophys J.    78:3260-3274.-   Tyagi, S. and F. R. Kramer. 1996. Molecular beacons: probes that    fluoresce upon hybridization. Nat. Biotechnol. 14:303-308.-   Vogelstein, B. and Kinzler, K. W. 1999. Digital PCR. Proc. Natl.    Acad. Sci. USA, 96, 9236-9241.-   Yuan, J., K. Matsumoto, and H. Kimura. 1998. A new tetradentate    beta-diketonate-europium chelate that can be covalently bound to    proteins for time-resolved fluoroimmunoassay. Anal. Chem.    70:596-601.-   Zuker, M. 2000. Calculating nucleic acid secondary structure. Curr.    Opin. Struct. Biol. 10, 303-310.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andExamples be considered as exemplary only. Those skilled in the art willrecognize, or will be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed by the following claims.

1. A composition comprising: (a) a stem-loop donor molecular beacon; and(b) a stem-loop acceptor molecular beacon, wherein resonance energytransfer occurs between the donor molecular beacon and the acceptormolecular beacon when the donor and acceptor molecular beacons hybridizeadjacent to each other on a target nucleic acid and are exposed to anexciting amount of electromagnetic radiation, and (c) means fordelivering the stem-loop donor molecular beacon and the stem-loop donormolecular beacons into a cell.
 2. The composition of claim 1, whereinthe donor molecular beacon comprises a resonance energy transfer donormoiety or a luminescence resonance energy transfer donor moiety.
 3. Thecomposition of claim 2, wherein the donor molecular beacon comprises aquencher that reduces emissions from the donor moiety when the donormolecular beacon is not hybridized to the target nucleic acid.
 4. Thecomposition of claim 2, wherein the donor moiety comprises afluorophore, Europium, Terbium, or a lanthanide chelate.
 5. Thecomposition of claim 1, wherein the acceptor molecular beacon comprisesa resonance energy transfer acceptor moiety or a luminescence resonanceenergy transfer acceptor moiety.
 6. The composition of claim 5, whereinthe acceptor moiety comprises a fluorophore or a phycobiliprotein. 7.The composition of claim 1, wherein acceptor molecular beacon comprisesa quencher that reduces emissions from the acceptor moiety when theacceptor molecular beacon is not hybridized to the target nucleic acid.8. The composition of claim 1, wherein emission from the acceptormolecular beacon produces a detectable signal resulting from resonanceenergy transfer.
 9. The composition of claim 1, wherein the energy toexcite the donor molecular beacon has a negligible capacity to directlyexcite the acceptor molecular beacon.
 10. The composition of claim 1,wherein unquenched emission from the donor moiety or acceptor moiety isdistinguishable from emissions from the acceptor moiety resulting fromresonance energy transfer.
 11. A composition comprising: (a) ashared-stem acceptor molecular beacon; and (b) a shared-stem donormolecular beacon, wherein fluorescence resonance energy transfer occursbetween the acceptor molecular beacon and the donor molecular beaconwhen the acceptor and donor molecular beacons hybridize adjacent to eachother on a target nucleic acid and are exposed to an exciting amount ofelectromagnetic radiation.
 12. The composition of claim 11, wherein thedonor molecular beacon comprises a resonance energy transfer donormoiety or a luminescence resonance energy transfer donor moiety.
 13. Thecomposition of claim 12, wherein the donor molecular beacon comprises aquencher that reduces emissions from the donor moiety when the donormolecular beacon is not hybridized to the target nucleic acid.
 14. Thecomposition of claim 12, wherein the donor moiety comprises afluorophore, Europium, Terbium, or a lanthanide chelate.
 15. Thecomposition of claim 11, wherein the acceptor molecular beacon comprisesa resonance energy transfer acceptor moiety or a luminescence resonanceenergy transfer acceptor moiety.
 16. The composition of claim 15,wherein the acceptor moiety comprises a fluorophore or aphycobiliprotein.
 17. The composition of claim 11, wherein acceptormolecular beacon comprises a quencher that reduces emissions from theacceptor moiety when the acceptor molecular beacon is not hybridized tothe target nucleic acid.
 18. The composition of claim 11, whereinemission from the acceptor molecular beacon produces a detectablesignal.
 19. The composition of claim 11, wherein the energy to excitethe donor molecular beacon has a negligible capacity to directly excitethe acceptor molecular beacon.
 20. The composition of claim 11, whereinunquenched emission from the donor moiety or acceptor moiety isdistinguishable from emissions from the acceptor moiety resulting fromresonance energy transfer.
 21. A method for detecting expression of atarget nucleic acid in a cell comprising: (a) delivering a stem-loopacceptor molecular beacon and a stem-loop donor molecular beacon to aninterior of a cell; (b) exposing the molecular beacons with an excitingamount of electromagnetic energy so that fluorescence resonance energytransfer occurs between the acceptor molecular beacon and the donormolecular beacon when the acceptor and donor molecular beacons hybridizeadjacent to each other on the target nucleic acid; and (c) detecting asignal resulting from fluorescence resonance energy transfer between thestem-loop donor molecular beacon and the stem-loop acceptor molecularbeacon in response to the exciting amount of electromagnetic energywherein a detectable signal indicates the presence of the target nucleicacid.
 22. The method of claim 21, wherein the target nucleic acidcomprises RNA.
 23. The method of claim 21, wherein the molecular beaconsare shared-stem molecular beacons.
 24. A method for detecting cancercomprising: (a) delivering a stem-loop acceptor molecular beacon and astem-loop donor molecular beacon to an interior of a cell suspected ofbeing cancerous, wherein the molecular beacons specifically hybridize toa subject nucleic acid that is differentially expressed in cancer cellscompared to non-cancer cells; (b) exposing the molecular beacons with anexciting amount of radiation so that fluorescence resonance energytransfer occurs between the acceptor molecular beacon and the donormolecular beacon when the acceptor and donor molecular beacons hybridizeadjacent to each other on the subject nucleic acid; and (c) detectingemissions from the acceptor molecular beacon resulting from resonanceenergy transfer, wherein a detectable signal is indicative of cancer.25. The method of claim 24, wherein the cancer is pancreatic cancer. 26.The method of claim 24, wherein the subject nucleic acid encodes anoncogene or tumor suppressor gene.
 27. The method of claim 26, whereinthe oncogene or tumor suppressor gene is selected from the groupconsisting of K-ras, p53, p16, MADH4, DPC4, BRCA2, MKK4, STK 11, TGFBR1,and TGFBR2.
 28. The method of claim 24, wherein the subject nucleic acidcan be used for the direct detection of an infectious disease agent orquantification of an infectious agent.
 29. The method of claim 24,wherein detectable emissions indicate the location the subject nucleicacid.
 30. The method of claim 24, wherein the molecular beacons areshared-stem molecular beacons.
 31. A method for identifying compoundsthat increase or decrease expression of a subject nucleic acidcomprising: (a) delivering stem-loop acceptor molecular beacons and astem-loop donor molecular beacons to an interior of a cell suspected ofbeing cancerous; (b) exposing the molecular beacons to an excitingamount of electromagnetic radiation so that fluorescence resonanceenergy transfer occurs between the acceptor molecular beacons and thedonor molecular beacons when the acceptor and donor molecular beaconshybridize adjacent to each other on the subject nucleic acid; (c)detecting a first signal from the acceptor molecular beacons resultingfrom resonance energy transfer; (d) contacting the cell comprising themolecular beacons with a test compound; (e) detecting a second signalfrom the acceptor molecular beacons resulting from resonance energytransfer, wherein in an increased second signal compared to the firstsignal indicates that the test compound increases expression of thetarget nucleic acid and a decreased second signal compared to the firstsignal indicates that the test compound decreases expression of thesubject nucleic acid.
 32. The method of claim 31, wherein the subjectnucleic acid encodes an oncogene or tumor suppressor gene.
 33. Themethod of claim 31, wherein the molecular beacons are shared-stemmolecular beacons.
 34. The method of claim 31, wherein the test compoundcomprises hormones or growth factors.
 35. A system for detecting atarget nucleic acid comprising: (a) a stem-loop donor molecular beacon;(b) a stem-loop acceptor molecular beacon, wherein resonance energytransfer occurs between the donor molecular beacon and the acceptormolecular beacon when the donor and acceptor molecular beacons hybridizeadjacent to each other on a target nucleic acid and are exposed to anexciting amount of electromagnetic radiation; (c) means for exciting themolecular beacons; (d) means for detecting emissions from the molecularbeacons; and (e) means for delivering the beacons inside the cell. 36.The system of claim 35, wherein the means for exciting the molecularbeacons comprises a laser, a one-photon excitation device, or atwo-photon excitation device.
 37. A kit comprising: a) a stem-loopacceptor molecular beacon that specifically hybridizes to a targetnucleic acid; b) a stem-loop donor molecular beacon that specificallyhybridizes to the target nucleic acid, wherein fluorescence resonanceenergy transfer occurs between the acceptor molecular beacon and thedonor molecular beacon when the acceptor and donor molecular beaconshybridize adjacent to each other on a target nucleic acid and areexposed to an exciting amount of electromagnetic radiation; and c)instructions for detecting the target nucleic acid using the molecularbeacons.
 38. The kit of claim 37, wherein the molecular beacons areshared-stem molecular beacons.
 39. The kit of claim 37, furthercomprising a means for delivering the molecular beacons into a cell.